KR20200134340A - Human igg1 fc region variants and uses thereof - Google Patents

Abstract

Polypeptides and related antibodies comprising a variant Fc domain are described herein. The variant Fc domain provides a stabilized Fc:Fc interaction when the polypeptide(s), antibody or antibodies bind to its target, antigen or antigens on the surface of the cell, thereby improving complement-dependent cytotoxicity (CDC ) Is provided.

Description

Human IgG1 Fc region variant and its use {HUMAN IGG1 FC REGION VARIANTS AND USES THEREOF}

The present invention relates to Fc domain-containing polypeptides, such as antibodies, that have increased complement-dependent cytotoxicity (CDC) resulting from one or more amino acid modifications in the Fc-domain, and may also have other modified effector functions.

Effector functions mediated by the Fc region of an antibody allow for the destruction of foreign substances, such as killing of pathogens and clearance and degradation of antigens. Antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are initiated by binding of the Fc region to Fc receptor (FcR)-bearing cells, whereas complement-dependent cytotoxicity (CDC) is initiated by binding of the Fc region to C1q, which initiates the classical pathway of complement activation.

Each IgG antibody contains two binding sites for C1q, one in each heavy chain constant (Fc) region. However, a single molecule of IgG in solution cannot activate complement (Sledge et al., 1973 J. Biol. Chem. 248, because the affinity of the monomeric IgG for C1q is very weak (K _d ~10 ^-4 M)). 2818-13; Hughes-Jones et al., 1979 Mol. Immunol. 16,697-701). Antigen-induced association of IgG can lead to much tighter binding (K _d ~10 ^-8 M) and complement activation of multivalent C1q molecules (Burton et al., 1990 Mol. Immunol. 22, 161-206). In contrast, IgM naturally exists as covalently linked pentamers or hexamers, and upon binding of cell expressed or immobilized antigens, IgM pentamers and hexamers can efficiently elicit CDCs. Antigen-binding is a requirement for inducing conformational changes in IgM to expose the C1q binding site (Feinstein et al., 1986, Immunology Today, 169-174).

In addition, it has been suggested that IgG can achieve complement activation by formation of a hexameric ring structure through the interaction of the CH2/CH3 domain of the Fc region (Burton et al., 1990 Trends in Biochem. Sci. 15, 64-64 69). Evidence supporting the existence of this hexameric IgG structure is two-dimensional (Reidler et al., 1986 I Handbook of Experimental Immunology 4 ^th edit.(Weir, DM ed.), pp17.1-17.5.Blackwell, Edinburgh; Pinteric et al. al., 1971 Immunochem. 8, 1041-5) and in three-dimensional crystals, as well as in IgG1, IgG2a and IgG4 and human Fc in solution (Kuznetsov et al., 2000 J Struct. Biol. 131, 108-115). . Hexameric ring formation was also observed in the crystal structure of the b12 human IgG1κ antibody directed against HIV-1 gp120 (1HZH in PDB) (Saphire et al., Science 2001 Aug 10;293(5532),1155-9). In the b12 hexameric ring, there are 6 accessible C1q binding sites on the hexamer surface, one from each of the 6 antibodies, with the other 6 binding sites facing down.

C1q is similar to a tulip bundle with 6 globular heads containing antibody binding regions, tethered to 6 collagenous stems (Perkins et al., 1985 Biochem J. 228, 13-26; Poon et al., 1983 J Mol Biol. 168, 563-77; Reid et al., 1983 Biochem Soc Trans 11, 1-12; Weiss et al., 1986 J. Mol. Biol. 189, 573-81). C1q fits well into the b12 hexamer assembly of the 1HZH crystal structure, and it was found that each of the six globular heads contacts one of the six C1q binding sites (Parren, FASEB Summer Research Conference, Snowmass, Co., 5- 10 July 2010; "Crystal Structure of an intact human IgG: implications for HIV-1 neutralization and effector function", Thesis by Erica Ollmann Saphire, for the Scripps Research Institute, La Jolla, California. November 2000). Mutations in selected amino acids in the Fc interface observed between symmetry-related b12 antibodies in the crystal structure were observed to decrease the avidity of C1q, indicating that these amino acids contribute to the intermolecular Fc:Fc interaction.

WO 2006/104989 describes modified antibody Fc regions and uses thereof.

WO 2005/047327 describes neonatal Fc receptor (FcRn)-binding polypeptide variants, dimeric Fc binding proteins, and methods associated therewith.

WO 2010/106180 describes Fc variants with increased binding to the neonatal Fc receptor (FcRn).

WO 2005/070963 describes polypeptide Fc region variants and uses thereof.

WO 2006/053301 describes Fc variants with altered binding to FcRn.

US 2011/0123440 describes altered antibody Fc-regions and uses thereof. The altered Fc-region has one or more amino acid substitutions.

US 2008/0089892 describes polypeptide Fc-region variants and compositions comprising such Fc-region variants.

US 2010/0184959 describes methods of providing Fc polypeptide variants with altered Fc ligand recognition and/or effector function.

US 2010/015133 describes a method of producing a polypeptide by modulating polypeptide association.

US 2010/105873 describes an integrated approach for generating multidomain protein therapeutics.

US 6,737,056 describes polypeptide variants with altered effector function.

Efforts have previously been made to identify antibody Fc-variants with enhanced effector function or other modified properties. Such studies include, for example, exchanging fragments between IgG isotypes for the production of chimeric IgG molecules (Natsume et al., 2008 Cancer Res 68(10), 3863-72) or within the hinge region (Dall'Acqua et al., 2006 J Immunol 177, 1129-1138) or in or near the C1q-binding site in the CH2 domain, concentrated around residues D270, K322, P329, and P331 (Idusogie et al., 2001 J Immunol 166, 2571-2575; Michaelsen et al., 2009 Scand J Immunol 70, 553-564 and WO 99/51642) focused on amino acid substitutions. See, for example, Moore et al. (2010 mAbs 2(2), 181-189)) describes testing various combinations of S267E, H268F, S324T, S239D, I332E, G236A and I332E for enhanced effector function via CDC or ADCC. Other Fc mutations that affect the binding to the Fc-receptor (WO 2006/105062, WO 00/42072, US Pat. No. 6,737,056 and US Pat. No. 7,083,784) or the physical properties of the antibody (WO 2007/005612 A1) have also been shown.

However, despite these and other advances in the art, there continues to be a need for new and improved antibody-based therapeutics.

The present invention provides polypeptide and antibody variants that have enhanced complement-dependent cytotoxicity (CDC) compared to their parental polypeptide/antibody and may also have other enhanced effector functions. Without being bound by theory, the variant is capable of more stable binding interactions between the Fc regions of the two polypeptide/antibody molecules, thereby having enhanced effector function, such as more avidity resulting in increased or more specific CDC responses. It is believed that a high surface is provided. Certain variants are also characterized by improved ADCC response, ADCP response, and/or other enhanced effector function. Such sophisticated mechanisms of polypeptide/antibody engineering can be applied to increase the efficacy or specificity of, for example, antibody-based therapeutics as described herein.

Thus, in one aspect, the present invention provides a parent polypeptide comprising an Fc domain and a binding region of an immunoglobulin at one or more amino acid residue(s) selected from the group corresponding to E430X, E345X and S440W in the Fc region of a human IgG1 heavy chain. It relates to a method of increasing the complement-dependent cytotoxicity (CDC) of said parental polypeptide comprising introducing a mutation of.

In a further aspect, the invention relates to a variant of a parental polypeptide comprising an Fc domain and a binding region of an immunoglobulin, wherein the variant is E430S, E430F, E430T, E345K, E345Q, E345R, E345Y in the Fc region of a human IgG1 heavy chain. , And one or more mutation(s) selected from the group corresponding to S440W, provided that the Fc domain does not contain any additional mutations that alter the binding of the variant to the neonatal Fc receptor (FcRn).

The invention also provides one or more such mutations for increasing complement-dependent cytotoxicity (CDC) mediated by a polypeptide or antibody, e.g., when bound to its antigen on the surface of an antigen-expressing cell, cell membrane or virion ( S).

In one aspect, a variant referred to herein as a “single-mutant” has an increased CDC compared to the parent polypeptide or antibody, and may also have other increased effector function.

In one aspect, a variant referred to herein as a "double-mutant" comprises at least two mutations in the segment and has an improved CDC compared to a variant comprising only one of the at least two mutations, It can also have other improved effector functions.

In one aspect, a variant referred to herein as a "mixed-mutant", when used in combination with a second variant of the same or different polypeptide or antibody comprising mutations in different amino acid residues within the fragment, Two variants, and the parental polypeptide or parental antibody alone, compared to one or more of the provided increased CDC, and may also have other increased effector function.

Typically, mutations are amino acid substitutions, such as the replacement of parent amino acid residues with residues of different sizes and/or physicochemical properties that promote the formation of new intermolecular Fc:Fc bonds or increase the strength of the interaction of an existing pair. It is a mutation. Exemplary amino acid residues for mutations according to the invention are shown in Tables 1 and 2A and B along with exemplary amino acid substitutions. A non-limiting description of different aspects of the invention is provided in FIG. 1.

Various uses and therapeutic applications for these and other aspects of the invention, particularly for polypeptide and antibody variants, are described in more detail below.

Figure 1: (a) Schematic representation of an IgG molecule forming a hexamer. Dotted circles show two adjacent Fc:Fc interaction pairs of two neighboring IgG molecules. The arrows in the box show the directions to look at the figures of b, c and d: the two neighboring Fc molecules rotate 90° (in the plane of the figure) and look in the direction of the CH3 domain from the Fab-arm. (b) Observed effect of oligomerization-enhancing mutations on CDC. Schematic representation showing Fc:Fc interaction pairs with increased efficacy according to the single mutant and double mutant aspects of the invention. (c) Observed effect of oligomerization-inhibiting mutations on CDC. In order to restore or increase the Fc:Fc interaction according to the double mutant and mixed mutant aspects of the present invention, how at least two oligomerization-inhibiting mutations complementary to each other are combined into one molecule (double mutant side ), or can be separated into two molecules (mixed mutant side). Mixed mutants achieve specific effector function activation that is dependent on the binding of the two antibodies, capable of recognizing different targets. (d) Theoretical effect of C1q binding-inhibiting mutations on CDC. Schematic representation of the Fc:C1q interaction, showing that if mutations inhibit C1q-binding, then these mutations cannot be combined or mixed to restore CDC activity because C1q cannot compensate for the defect introduced in the antibody.
Figure 2: Human IgG1, IgG1f, IgG2, IgG3, IgG4, IgA1, IgA2 corresponding to residues P247 to K447 in the IgG1 heavy chain numbered by the EU index as shown in Kabat, using Cluster 2.1 software. , IgD, IgE and IgM Fc fragment sequence alignment. The sequence shown is a human IgG1 heavy chain constant region (SEQ ID NO: 1; Uniprot accession number P01857) and residues 130 to 330 of the allogeneic variant IgG1m(f); Residues 126 to 326 of the IgG2 heavy chain constant region (SEQ ID NO: 2; Uniprot accession number P01859); And residues 177 to 377 of the IgG3 heavy chain constant region (SEQ ID NO: 2; Uniprot accession number P01860); And residues 127 to 327 of the IgG4 heavy chain constant region (SEQ ID NO: 4; Uniprot accession number P01861); And residues 225-428 of the IgE constant region (Uniprot Accession No. P01854); And residues 133-353 of the IgA1 constant region (Uniprot Accession No. P01876); And residues 120-340 of the IgA2 constant region (Uniprot Accession No. P01877); And residues 230-452 of the IgM constant region (Uniprot Accession No. P01871); And residues 176-384 of the IgD constant region (Uniprot Accession No. P01880).
3A and B: Sequence alignment of anti-EGFr antibody 2F8 in the IgG1 (SEQ ID NO: 3), IgG4 (SEQ ID NO: 5) and (partially) IgG3 (SEQ ID NO: 6) backbones. Amino acid numbering according to Kabat and EU-index is shown (both by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Described in).
Figure 4: K439/S440 between the Fc (Fc and Fc', respectively) of adjacent molecules in a multimeric (e.g., hexameric) arrangement, showing the interaction between wild-type, unmodified Fc and Fc' molecules. Detailed view of the interaction.
Figure 5: Between the Fc (respectively, Fc and Fc') of adjacent molecules in a multimeric (e.g., hexameric) arrangement, showing the interaction between variant Fc and Fc' molecules comprising K439E and S440K mutations Detailed view of the K439/S440 interaction.
Figure 6: C1q binding ELISA using 7D8 Fc:Fc mutant. A series of concentrations of the indicated antibody were coated into the wells of a microtiter plate and incubated with a fixed concentration of C1q. The efficiency of binding to C1q in all coated mutants except I253D was comparable to that of wild type 7D8. At least three representative experiments are presented.
Figure 7: CDC mediated by the 7D8 variant against CD20-positive Raji cells. Raji cells were incubated with 7D8 mutants (K439E, S440K, K439E/S440K double mutants, K439E + S440K mixture) and a series of concentrations of C1q to test CDC efficacy by measuring cell lysis. Representative graphs of repeated experiments are presented.
Figure 8: CDC mediated by the 7D8 mutant (7D8-WT, K439E, S440K, K439E/S440K double mutant, K439E + S440K mixture) against CD20-positive Daudi cells. A series of concentrations of the 7D8 mutants were tested for their efficacy in inducing CDC.
Figure 9: CDC mediated by mutants of the CD38 antibody HuMAb 005 against CD38-positive cells. (a) CDC efficacy against Daudi cells by a series of concentrations of 005 mutants. (b) CDC efficacy on Raji cells with a series of concentrations of HuMAb 005 mutants. (c) CDC efficacy of the E345R mutant of HuMAb 005 using either 20% or 50% NHS on Wien133 cells. (d) CDC efficacy of E345R mutants of HuMAb 005 and 7D8 using either 20% or 50% NHS on Raji cells. Crude antibody samples isolated from transient transfection were tested. As a negative control, the supernatant of mock-transfected cells was used.
Figure 10: CDC by wild type and E345R mutants of CD38 antibody HuMAb 005 (a) and CD20 antibody HuMAb 7D8 (b) in a competition experiment using an Fc-binding peptide. Cell lysis was measured after CDC against antibody-opsonized Daudi-cells incubated with a series of concentrations of Fc-binding DCAWHLGELVWCT peptide (SEQ ID NO: 7). Crude antibody samples isolated from transient transfection were used. As a negative control, the supernatant of mock-transfected cells was used.
Figure 11: ADCC of CD38 expressing Daudi cells by wild-type CD38 antibody HuMAb 005 and mutant IgG1-005-E345R. The ADCC of one donor's PBMC is shown (shown as% lysis).
Figure 12: Binding of wild type IgG1-7D8 and mutant IgG1-7D8-E345R to human, cynomolgus and mouse FcRn, as determined by ELISA at pH 6.
Figure 13: Plasma concentrations of wild-type IgG1-7D8 and -E354R, -S440K and K322A variants after intravenous injection in SCID mice.
14A, B, C, and D: CDC against CD20- and CD38-positive Wien133 cells.
15A and B: Evaluation of the in vivo efficacy of IgG1-7D8-E345R in a subcutaneous xenograft model using Raji-luc #2D1 cells.
16A and B: Evaluation of the in vivo efficacy of IgG1-005-E345R in a subcutaneous xenograft model using Raji-luc #2D1 cells.
Figure 17: CDC against CD38-positive, EGFR-negative Wien133 cells by CD38/EGFR bispecific antibody with E345R mutation.
18A and B: CDC against CD20-positive, CD38-negative Wien133 cells or Raji cells with CD20/CD38 bispecific antibodies with and without E345R mutation.
Figure 19: CDC against EGFR-positive A431 cells by EGFR antibody 2F8 with E345R mutation.
20A and B: CDC mediated by the E345R mutant antibody.
Figure 21: Analysis of co-localization of lysosome marker LAMP1 (APC) and TF antibody (FITC).
22A-D: Introduction of E345R results in enhanced CDC-mediated killing compared to wild-type rituximab tested against different B cell lines.
Figure 22E: The introduction of E345R increased compared to wild-type rituximab, regardless of the expression levels of complement regulatory proteins CD46 (a), CD55 (b) or CD59 (c) in different B cell lines with comparable CD20 expression levels. Causes maximum CDC-mediated death.
Figure 23: CDC kinetics. The E345R antibody results in faster and more substantial lysis of target cells by CDC compared to wild-type antibodies.
Figure 24: CDC kinetics. Introducing the E345R mutation in the bispecific CD38xCD20 antibody results in faster and more substantial CDC-mediated target cell lysis.
25A-B: CDC kinetics. Introducing the E345R mutation to the bispecific antibodies CD38xEGFR (a) and CD20xEGFR (b) that monovalently binds to EGFR-negative Raji cells results in faster and more substantial CDC-mediated target cell lysis.
Figures 26A-F: CDC against Wien133 cells by a combination of mutant and wild-type antibodies containing (ac) E345R and Q386K or (df) E345R, E430G and Q386K. The IgG1-b12 mutant did not bind to Wien133 cells and was used as a negative control antibody.
Figure 27: CDC efficacy of IgG1, IgG2, IgG3, and IgG4 isotype antibodies containing the E345R mutation.
Figure 28: Introduction of the Fc-Fc stabilized E345R mutation to wild-type CD38 antibody 005 results in enhanced killing of primary CLL cells in an ex vivo CDC assay (mean±standard error of the mean).
Figure 29: FcRn binding of wild-type IgG1-005 and IgG1-005 mutants to human, mouse, and cynomolgus FcRn at pH 6.0, as determined by ELISA.
Figure 30: CDC efficacy in 20% normal human serum of various rituximab mutants, wild type rituximab and unrelated negative control antibody IgG1-b12 in Ramos and SU-DHL-4 cell lines.
Figure 31: Wild type IgG1-005, IgG1-005-E345K, IgG1-005-E345Q, IgG1-005-E345Y, IgG1-005-E430G, IgG1- as determined by Micro Vue C4d-fragment ELISA C4d production in normal human serum of 005-E430S, and IgG1-005-S440Y, and heat aggregated IgG (HAG) (positive control).
Figure 32A/B: Administered wild-type IgG1-005 and antibody variants IgG1-005-E345K, IgG1-005 in SCID mice as determined by total human IgG ELISA (Figure 32A) and human CD38 specific ELISA (Figure 32B). Plasma clearance rates of -E345Q, IgG1-005-E345R, IgG1-005-E345Y, IgG1-005-E430F, IgG1-005-E430G, IgG1-005-E430S, IgG1-005-E430T, and IgG1-005-S440Y.

As described herein, surprisingly, mutations in amino acids that are not directly involved in Fc:C1q binding can nonetheless increase the CDC of the antibody and may also improve other Fc-mediated effector functions of the antibody. This supports the hypothesis that antibody molecules, such as IgG1 antibodies, can later form oligomeric structures that are bound by Clq. In addition, some mutations were found to reduce CDC-induction, but the combination of some of these mutations in the same or different antibody molecules led to CDC-induced recovery, and showed additional specificity for oligomerization of the antibody, and thus more It promoted specific CDC-induction. In addition, certain mutations that increase the CDC-response were characterized by improved ADCC responses, increased avidity, increased internalization and in vivo efficacy in the mouse tumor model system as shown in the Examples. These findings allow the development of new antibody-based therapeutics with enhanced CDC-inducing ability, more selective CDC-induction, and/or other improved effector function.

The polypeptide variants (including antibody variants) of the present invention comprising one or more mutation(s) in the fragment corresponding to amino acid residues E345 to S440 in IgG1 all comprise a binding region and a full-length or partial Fc domain of an immunoglobulin. Without being bound by theory, the mutations identified are schematically presented in FIG. 1 and are based on three different theories referred to herein as “single mutants”, “double mutants” and “mixed mutants” and It is believed to result in a more specific CDC-induction.

The improved C1q and/or CDC effects of the variants of the invention are mainly detectable only in assays that allow antibody oligomer formation, such as cell-based assays in which the antigen is not immobilized and is present in a fluid membrane. In addition, it can be confirmed according to the theory presented in Fig. 1c that this effect arises from more stable antibody oligomers, not from modification of the direct binding site of C1q.

Justice

The term “single-mutant” should be understood as a variant of the invention that has an increased CDC compared to the parent polypeptide or antibody and may also have other enhanced effector functions.

The term "double-mutant" is a variant comprising at least two mutations in the segment, has improved CDC compared to a variant comprising only one of said at least two mutations, and also exhibits other enhanced effector functions. It should be understood as a possible variant.

The term “mixed-mutant”, when used in combination with a second variant of the same or different polypeptide or antibody comprising mutations in different amino acid residues within the segment, is a variant, a second variant, and a parent polypeptide or antibody alone. It should be understood as a variant that provides increased CDC compared to one or more, and optionally other enhanced effector functions.

The term "polypeptide comprising the Fc-domain and binding region of an immunoglobulin" in the context of the present invention refers to the Fc-domain of an immunoglobulin, and any molecule present on, for example, a cell, a bacterium, or a virion, For example, it refers to a polypeptide comprising a binding region capable of binding to a polypeptide. The Fc-domain of an immunoglobulin comprises two CH2-CH3 regions of an immunoglobulin and a linking region, e.g., a hinge region, which is usually produced after digestion of the antibody by papain (known to the skilled person). It is defined as a fragment of an antibody. The constant domain of the antibody heavy chain defines the antibody isotype, for example IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD or IgE. The Fc-domain mediates the effector functions of proteins and antibodies of cell surface receptors and complement systems called Fc receptors. The binding region may be a polypeptide sequence capable of binding cells, bacteria or virions, such as proteins, protein ligands, receptors, antigen-binding regions, or ligand-binding regions. If the binding region is, for example, a receptor, a "polypeptide comprising an Fc-domain of an immunoglobulin and a binding region" can be prepared as a fusion protein of the Fc-domain of an immunoglobulin and the binding region. If the binding region is an antigen-binding region, the "polypeptide comprising the Fc-domain of an immunoglobulin and the binding region" may be an antibody, such as a chimeric, humanized or human antibody, or a heavy chain alone antibody or ScFv-Fc-fusion. Polypeptides comprising the Fc-domain and binding region of an immunoglobulin may typically comprise a linking region, e.g., a hinge region, and the two CH2-CH3 regions of the heavy chain of an immunoglobulin, and thus "immunoglobulin A polypeptide comprising the Fc-domain and binding region of a globulin" may be "a polypeptide comprising at least the Fc-domain and binding region of an immunoglobulin". The term "Fc-domain of an immunoglobulin" in the context of the present invention refers to a linking region, for example a subtype of antibody, for example human IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgM or IgE. It means that the hinge, and the CH2 and CH3 regions of the immunoglobulin are present. The polypeptide is not limited to human origin, but can be of any origin, such as, for example, of mouse or cynomolgus origin.

The term “CH2 region” or “CH2 domain” as used herein is intended to refer to the CH2 region of an immunoglobulin. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 228-340 according to the EU numbering system. However, the CH2 region can also be of any other subtype as described herein.

The term “CH3 region” or “CH3 domain” as used herein is intended to refer to the CH3 region of an immunoglobulin. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the EU numbering system. However, the CH3 region can also be of any other subtype as described herein.

The term "immunoglobulin" is a structurally related glycoprotein consisting of two pairs of polypeptide chains, a pair of low molecular weight light chains (L) and a pair of heavy chains (H), all four potentially interconnected by disulfide bonds. Indicates the class of The structure of immunoglobulins is widely characterized. See, eg, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain is typically composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region typically consists of three domains, CH1, CH2 and CH3. The heavy chains are interconnected through disulfide bonds in the so-called "hinge region". Each light chain is typically composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically consists of one domain CL. VH and VL regions can be further subdivided into hypervariable regions, also called complementarity determining regions (CDRs) (or hypervariable regions whose sequences are hypervariable and/or can form structurally defined loops), and these regions There are scattered more conserved areas called framework areas (FR). Each VH and VL typically consists of 3 CDRs and 4 FRs arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901 917 (1987)). Unless otherwise indicated or contradicted by context, amino acids of the constant region sequence are numbered according to the EU-index herein (Kabat, EA et al., Sequences of proteins of immunological interest. 5th Edition-US Department of Health and Human Services, NIH publication No. 91-3242, pp 662,680,689 (1991)).

The term “antibody” (Ab) in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either of these, which under typical physiological conditions is at least about 30 minutes, at least about 45 Minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, A significant period of time, such as 7 days or more, etc., or any other relevant time that is functionally defined (e.g., induces, promotes, and/or enhances a physiological response associated with antibody binding to an antigen and/or It has the ability to specifically bind to an antigen with a half-life of sufficient time to cause or modulate and/or sufficient time for the antibody to mobilize effector activity). The antibody of the present invention comprises the Fc-domain and antigen-binding region of an immunoglobulin. Antibodies generally comprise two CH2-CH3 regions and a linking region, such as a hinge region, such as at least an Fc-domain. Thus, the antibody of the present invention may comprise an Fc region and an antigen-binding region. The variable regions of the heavy and light chains of immunoglobulin molecules contain binding domains that interact with antigens. The constant or “Fc” region of an antibody is an immunoglobulin for host tissues or factors (including various cells of the immune system (eg, effector cells)) and components of the complement system, such as C1q (the first component of the classical pathway of complement activation). Can mediate the combination of Antibodies can also be multispecific antibodies, such as bispecific antibodies or similar molecules. The term “bispecific antibody” refers to an antibody having specificity for at least two different epitopes, typically non-overlapping epitopes. These epitopes can be on the same or different targets. When epitopes are on different targets, these targets can be on the same cell or on different cells or cell types. As indicated above, the term antibody herein includes fragments of antibodies that contain at least a portion of the Fc-region and retain the ability to specifically bind to an antigen, unless otherwise indicated or clearly contradicted by context. Such fragments can be provided by any known technique, such as enzymatic cleavage, peptide synthesis and recombinant expression techniques. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "Ab" or "antibody" are monovalent antibodies (WO2007059782 (described in Genmab); consisting of only two heavy chains, for example heavy chain antibodies naturally occurring in camels ( For example, Hamers-Casterman (1993) Nature 363:446); ThioMab (Roche, WO2011069104), a symmetrical and bispecific antibody-like molecule, a strand-exchange engineered domain (SEED or Seed-body) (Merck, WO2007110205); Triomab (Fresenius, Lindhofer et al. (1995 J Immunol 155:219))); FcΔAdp ( Regeneron, WO2010151792), azimetric scaffold (Zymeworks/Merck, WO2012/058768), mAb-Fv (Xencor, WO2011/028952), double variable domain immunoglobulin ( Abbott, DVD-Ig, U.S. Patent No. 7,612,181); dual domain double head antibody (Unilever; Sanofi Aventis, WO20100226923), Di-diabody (ImClone) /Eli Lilly), Knobs-into-holes antibody format (Genentech, WO9850431); DuoBody (Genmab, WO 2011/131746); Electrostatic steering Antibody format (Amgen, EP1870459 and WO 2009089004; Chugai, US201000155133; Oncomed, WO2010129304A2); Bispecific IgG1 and IgG2 (Rinat neurosciences Corporation, WO11143545 ), cross MAb (Roche, WO2011117329), LUZ-Y (Genentech), Biclonic (Merus), dual targeting domain antibody (GSK/Domantis), two-in-one recognizing two targets (Two-in-one) antibody (Genentech, NovImmune), bridging Mab (Karmanos Cancer Center), CovX-body (CovX/Pfizer), IgG-like bispecific Red (Imclohn/Ely Lily, Shen, J., et al. J Immunol Methods, 2007. 318(1-2): p. 65-74]), and DIG-body and PIG-body (Pharmabcine), and a dual-affinity retargeting molecule (Fc-DART or Ig-DART, Macrogenics, WO/2008/157379 , WO/2010/080538), Zybodies (Zyngenia), a common light chain (Crucell/Merus, US7262028) or a common heavy chain (κλ body, Novimune) Fusion protein comprising a polypeptide sequence fused to an antibody fragment containing an Fc-domain-like scFv-fusion as well as an Fc-domain-like scFv-fusion protein (same as BsAb, ZymoGenetics/BMS), HERCULES (Biogen IDec ( Biogen Idec)) (US007951918), SCORPIONS (Emergent BioSolutions/Trubion), Ts2Ab (MedImmune)/AZ (Dimasi, N., et al. J Mol Biol , 2009. 393(3): p. 672-92), scFv fusion (Novartis), scFv fusion (Changzhou Adam Biotech Inc) (CN 102250246), TvAb (Roche) (WO 2012025525, WO 2012025530), mAb ² (f-Star) (WO2008/003116), and dual scFv-fusions. Further, unless otherwise specified, the term antibody refers to the term antibody. Polyclonal antibodies, monoclonal antibodies (e.g., human monoclonal antibodies), e.g., antibody mixtures produced by techniques used by Sympogen and Merus (Oligoclonics) ( Recombinant polyclonal), and antibody-like polypeptides such as chimeric antibodies and humanized antibodies. The resulting antibodies potentially have any isotype. I can.

The term “full length antibody” as used herein refers to an antibody (eg, parental or variant antibody) containing all heavy and light chain constant and variable domains corresponding to those commonly found in isotype wild-type antibodies.

The term “human antibody” as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the present invention are amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo, insertion Or deletion). However, the term “human antibody” as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as mice, have been grafted to human framework sequences.

As used herein, the terms “monoclonal antibody”, “monoclonal Ab”, “monoclonal antibody composition”, “mAb” and the like refer to a preparation of Ab molecules of single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope. Thus, the term “human monoclonal antibody” refers to an Ab that exhibits single binding specificity, having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs are obtained from transgenic or transchromosomal non-human animals, such as transgenic mice, having a genome comprising a human heavy chain transgene repertoire and a light chain transgene repertoire, rearranged to produce functional human antibodies and fused to immortalized cells. It can be produced by hybridomas containing B cells.

As used herein, "isotype" refers to the class of immunoglobulins encoded by heavy chain constant region genes (eg, IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE, or IgM or any allotype thereof. , IgG1m(za) and IgG1m(f)). In addition, each heavy chain isoform can be combined with a kappa (κ) or lambda (λ) light chain.

The term "monovalent antibody" in the context of the present invention means that the antibody molecule is capable of binding to an antigen only by one of the binding domains of the antibody, for example having a single antigen-antibody interaction, and thus crosslinking to the antigen. Means there is no.

As used herein, the term “target” should be understood as a molecule to which the binding region of a polypeptide comprising an Fc domain and a binding region binds in the context of the present invention, and in terms of binding of an antibody, when used, the resulting antibody is indicated. Includes any antigen. The terms “antigen” and “target” may be used interchangeably with respect to an antibody and, in any aspect or embodiment of the invention, may constitute the same meaning and purpose.

As used herein, the term "binding" in the context of binding of an antibody to a given antigen is typically used in a BIAcore 3000 instrument, for example using the antigen as a ligand and the antibody as an analyte. About 10 ^-6 M or less, such as 10 ^-7 M or less, such as about 10 ^-8 M or less, such as about 10 ^-9 M or less, about 10 ^-10 M or less, as determined by surface plasmon resonance (SPR) technique , Or a binding having an affinity corresponding to a K _D of about 10 ^-11 M or even less, and to a given antigen or a non-specific antigen other than a closely related antigen (e.g., BSA, casein). Binding to a given antigen with an affinity corresponding to a K _D that is at least 10 times lower, such as at least 100 times lower, such as at least 1,000 times lower, such as at least 10,000 times lower, for example at least 100,000 times lower than its binding affinity for do. The amount that exhibits a lower affinity is dependent on the K _D of the antibody, and thus, if the K _D of the antibody is very low (i.e., the antibody is highly specific), then for an antigen that is lower than the affinity for a non-specific antigen. The amount representing affinity may be at least 10,000 times. The term “K _D ” (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

A "variant" or "antibody variant" or "variant of a parent antibody" of the present invention is an antibody molecule comprising one or more mutations compared to a "parent antibody". Different terms may be used interchangeably and, in any aspect or embodiment of the present invention, may constitute the same meaning and purpose. Exemplary parental antibody formats include, but are not limited to, wild type antibodies, full length antibodies or Fc-containing antibody fragments, bispecific antibodies, human antibodies, or any combination thereof. Similarly, a "variant" of the present invention or "a variant of a polypeptide comprising an Fc-domain of an immunoglobulin and a binding region" or "a variant of a parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region" It is a "polypeptide comprising the Fc-domain and binding region of an immunoglobulin" comprising one or more mutations compared to "a parent polypeptide comprising the Fc-domain and binding region of an immunoglobulin". Different terms may be used interchangeably and, in any aspect or embodiment of the present invention, may constitute the same meaning and purpose. Exemplary mutations include amino acid deletions, insertions, and substitutions of amino acids within the parent amino acid sequence. Amino acid substitutions can exchange a natural amino acid for another naturally occurring amino acid, or for a non-naturally occurring amino acid derivative. Amino acid substitutions can be conservative or non-conservative substitutions. In the context of the present invention, conservative substitutions may be defined by substitutions within a class of amino acids reflected in one or more of the following three tables:

In the context of the present invention, substitutions in variants are denoted as follows:

Original amino acid-position-substituted amino acid;

Three letter codes, or one letter codes, including the codes Xaa and X are used to represent amino acid residues. Thus, the expression “E345R” or “Glu345Arg” means that the variant comprises a substitution of glutamic acid for arginine at the variant amino acid position corresponding to the amino acid at position 345 in the parent antibody.

Even if the position is not present in the antibody, variants include, for example, insertions of the following amino acids:

Position-substituted amino acid; The expression, for example "448E" is used.

This expression is particularly relevant to the modification(s) in a series of homologous polypeptides or antibodies.

Similarly, if the type of substitution amino acid residue(s) is not important:

Original amino acid-position; Or "E345".

In the case of modifications in which the original amino acid(s) and/or substituted amino acid(s) may contain more than one amino acid, but not all amino acid(s), substitution of glutamic acid with arginine, lysine or tryptophan at position 345 :

"Glu345Arg,Lys,Trp" or "E345R,K,W" or "E345R/K/W" or "E345 → R, K or W"

Can be used interchangeably in the context of the present invention.

In addition, the term “substitution” includes substitution with any one of the other 19 natural amino acids, or with another amino acid, such as a non-natural amino acid. For example, the substitution of amino acid E at position 345 includes each of the following substitutions: 345A, 345C, 345D, 345G, 345H, 345F, 345I, 345K, 345L, 345M, 345N, 345Q, 345R, 345S, 345T. , 345V, 345W, and 345Y. This is equivalent to the expression 345X, where X represents any amino acid. These substitutions may also be designated E345A, E345C, etc. or E345A,C, etc., or E345A/C/etc. The same manner applies similarly to each and every position mentioned herein, especially so that any one of these substitutions is included herein.

An amino acid or segment in one sequence that "corresponds to" an amino acid or segment in another sequence is (i) another amino acid or segment using a standard sequence alignment program, such as ALIGN, Cluster W or the like, typically in default settings And (ii) at least 50%, at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO:1. For example, the sequence alignments shown in Figures 2 and 3 can be used to identify any amino acid in the IgG2, IgG3 or IgG4 Fc sequence that corresponds to a specific amino acid in the IgG1 Fc sequence.

The present invention relates to variants having a certain degree of identity to amino acids P247 to K447 of SEQ ID NOs: 1, 2, 3, 4, and 5, i.e. parental polypeptides and parental antibodies, and/or variant polypeptides and variant antibodies, And/or variant antibodies are hereinafter referred to as “homologous antibodies”.

For the purposes of the present invention, the degree of identity between the two amino acid sequences, as well as the degree of identity between the two nucleotide sequences, is determined by the program "align" which is a Needleman-Wunsch alignment (ie, global alignment). Determined by The program is used for alignment of nucleotide sequences as well as polypeptides. The default scoring matrix BLOSUM50 is used for polypeptide alignment, the default identity matrix is used for nucleotide alignment, and the penalty of the first residue in the gap is -12 for the polypeptide and -16 for the nucleotide. The penalty for additional residues in the gap is -2 for polypeptides and -4 for nucleotides.

“Align” is part of the FASTA package version v20u6 (WR Pearson and DJ Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448, and WR Pearson (1990) “Rapid and Sensitive Sequence. Comparaison with FASTP and FASTA", Methods in Enzymology 183:63-98). FASTA protein alignment uses the Smith-Waterman algorithm, which does not impose restrictions on the gap size ("Smith-Waterman algorithm", TF Smith and MS Waterman (1981) J. Mol. Biolo. 147:195 -197]).

As used herein, the term “effector cell” refers to an immune cell that is involved in the effector stage of the immune response, as opposed to the stage of recognition and activation of the immune response. Exemplary immune cells are cells of bone marrow or lymph origin, such as lymphocytes (e.g., B cells and T cells (including cytolytic T cells (CTL))), killer cells, natural killer cells, macrophages, monocytes, eosinophils, Polymorphonuclear cells such as neutrophils, granulocytes, mast cells and basophils. Some effector cells express an Fc receptor (FcR) or complement receptor and perform certain immune functions. In some embodiments, effector cells, such as, for example, natural killer cells, are capable of inducing ADCC. For example, monocytes, macrophages, neutrophils, dendritic cells, and Kupffer cells expressing FcRs have specific killing of target cells and presentation of antigens to other components of the immune system, or binding to antigen-presenting cells. Get involved in In some embodiments, ADCC can be further enhanced by antibody-induced classical complement activation that results in the deposition of activated C3 fragments on target cells. The C3 cleavage product is a ligand for the complement receptor (CR) expressed on bone marrow cells, such as CR3. Recognition of complement fragments by CR on effector cells can promote enhanced Fc receptor-mediated ADCC. In some embodiments, antibody induced classical complement activation induces C3 fragments onto target cells. These C3 cleavage products can promote direct complement-dependent cellular cytotoxicity (CDCC). In some embodiments, effector cells are capable of phagocytosing a target antigen, target particle or target cell. Expression of specific FcR or complement receptors on effector cells can be regulated by humoral factors such as cytokines. For example, expression of FcγRI has been found to be upregulated by interferonγ (IFNγ) and/or G-CSF. This enhanced expression increases the cytotoxic activity of FcγRI-bearing cells on the target. Effector cells are capable of phagocytosis or lysis of a target antigen or target sequence. In some embodiments, antibody induced classical complement activation induces C3 fragments onto target cells. These C3 cleavage products can promote phagocytosis directly by effector cells or indirectly by enhancing antibody mediated phagocytosis.

As used herein, the term “vector” is intended to refer to a nucleic acid molecule capable of inducing the transcription of a nucleic acid segment ligated into the vector. One type of vector is a "plasmid", which is in the form of a circular double stranded DNA loop. Another type of vector is a viral vector, in which nucleic acid segments can be ligated into the viral genome. Certain vectors are capable of self-replicating within the host cell into which they have been introduced (eg, bacterial vectors and episomal mammalian vectors having a bacterial origin of replication). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell, and thus replicate with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”). In general, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably, because plasmid is the most commonly used form of vector. However, the present invention is intended to include other types of expression vectors that perform equivalent functions, such as viral vectors (eg, replication-deficient retroviruses, adenoviruses and adeno-associated viruses).

The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which an expression vector has been introduced. It is to be understood that the term is intended to refer not only to a particular cell of interest, but also to the progeny of that cell. Because certain modifications may occur in the next generation due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. Recombinant host cells include, for example, transfectomas such as CHO cells, HEK-293 cells, PER.C6, NS0 cells, and lymphocyte cells, and prokaryotic cells such as E. E. coli and other eukaryotic hosts, such as plant cells and fungi.

As used herein, the term “transfectoma” refers to a recombinant eukaryotic host cell expressing an Ab or a target antigen, such as CHO cells, PER.C6, NS0 cells, HEK-293 cells, plant cells, or fungi, such as yeast cells. Includes.

The term “agent” refers to an increased ability to form oligomers when interacting with an antigen associated with a cell, cell membrane, virion or other structure (eg, an antigen expressed on the surface of a cell), thereby increasing C1q binding. , Complement activation, CDC, ADCC, ADCP, other Fc-mediated effector functions, internalization, downregulation, apoptosis, antibody-drug-conjugate (ADC) uptake, avidity, or any combination thereof, allowing antibody variants and different antibody variants Means a mixture of. Exemplary assays include, in the examples, for example C1q-binding strength (Example 4), CDC (Examples 5, 6 and 10, 16, 19, 22, 23, 24, 25, and 35); ADCC (Example 12), in vivo efficacy (Example 20, 21), plasma clearance rate (Example 37), FcRn binding (Example 34), and target independent fluid phase complement activation (Example 36) Is presented. Variants according to aspects of the present application referred to herein as “single-mutants”, “dual-mutants”, and “mixed-mutants” are described in further detail below along with exemplary preparations and methods of use thereof. .

As used herein, the term “affinity” refers to the binding of one molecule, eg, to another molecule of an antibody, eg, a target or antigen, at a single site, eg, monovalent binding of an individual antigen binding site of an antibody to an antigen. Is the strength of.

The term “avidity” as used herein refers to the combined strength of multiple binding sites between two constructs, such as between multiple antigen binding sites of an antibody that interact simultaneously with a target, or between an antibody and C1q, for example. If there is more than one binding interaction, the two structures will dissociate only when all of the binding sites are dissociated, so the rate of dissociation will be slower than at the individual binding sites, and thus the binding strength (affinity) of the individual binding sites Will provide a greater effective total bond strength (bonding force) compared to.

As used herein, the term “oligomer” refers to a molecule (eg, an antibody) consisting of more than one but limited number of monomer units, at least in principle compared to a polymer consisting of an unrestricted number of monomers. Exemplary oligomers are dimers, trimers, tetramers, pentamers and hexamers. The Greek prefix is often used to designate the number of monomer units in an oligomer, for example a tetramer consists of 4 units and a hexamer consists of 6 units.

As used herein, the term “oligomerization” is intended to refer to the process of converting a monomer to a limited degree of polymerization. It is observed herein that oligomerization of the Fc-domain preferably occurs after target binding by an Fc-domain containing polypeptide, such as an antibody, on the cell surface (but not limited thereto). Oligomerization of antibodies can be performed, for example, by cell surface C1q-binding assay (as described in Examples 4 and 9), C1q potency assay (as described in Example 5) and complement dependent cytotoxicity (Examples 6, 10 and 19) can be used.

The term "C1q binding" as used herein is intended to refer to the binding of C1q in the context of the binding of C1q to an antibody bound to its antigen. Antibodies bound to their antigens should be understood to occur both in vivo and in vitro in the context as described herein. Clq binding, for example, using an antibody immobilized on an artificial surface (e.g., plastic of an ELISA plate as described in Example 3) or using an antibody bound to a given antigen on the surface of a cell or virion. Can be evaluated (as described in Examples 4 and 9). Binding of C1q to an antibody oligomer should be understood herein as a multivalent interaction resulting in high avid binding.

The term “complement activation” as used herein refers to the activation of the classical complement pathway triggered by the binding of the complement component C1q to an antibody bound to its antigen. C1q is the first protein of the early event of the classical complement cascade, which involves a series of cleavage reactions that terminate with the active formation of an enzyme called C3 convertase, which cleaves complement component C3 into C3b and C3a. C3b covalently binds to C5 on the membrane to form C5b, which in turn triggers a later event of complement activation in which the terminal complement components C5b, C6, C7, C8 and C9 are associated with the membrane attack complex (MAC). The complement cascade produces pores that cause cell lysis, also known as complement-dependent cytotoxicity (CDC). Complement activation was determined by C1q efficacy (as described in Example 5), CDC kinetics (as described in Examples 28, 29, and 30), CDC assays (Examples 6, 10, 19, 25, 27, 33, and 35) or Beurskens et al. April 1, 2012 vol. 188 no. 7 3532-3541] can be evaluated by the cell deposition method of C3b and C4b.

As used herein, the term “complement-dependent cytotoxicity” (“CDC”) is an antibody-mediated complement that results in the lysis of an antibody bound to its target on a cell or virion as a result of pores in the membrane produced by MAC assembly. It is intended to refer to the process of activation. CDC is an in vitro assay as described in Examples 6, 10, 19, 25, 27, 33, and 35, such as a CDC assay in which normal human serum is used as the complement source, or a C1q potency assay as described in Example 5. (Here, normal human serum is limited at C1q).

The term “antibody-dependent cell-mediated cytotoxicity” (“ADCC”) as used herein refers to the mechanism of death of antibody-coated target cells or virions by cells expressing an Fc receptor that recognizes the constant region of the bound antibody. It is intended to refer to. ADCC can be determined, for example, using a method such as the ADCC assay described in Example 12.

The term “antibody-dependent cellular phagocytosis” (“ADCP”) as used herein is intended to refer to the mechanism of removal of antibody-coated target cells or virions by internalization by phagocytic cells. The internalized antibody-coated target cells or virions are contained in vesicles called phagosomes, which are then fused with one or more lysosomes to form phagolysosomes. ADCP is an in vitro cytotoxicity assay using macrophages as effector cells and described in van Bij et al. in Journal of Hepatology Volume 53, Issue 4, October 2010, Pages 677-685, or using, for example, S. Aureus (S. aureus) phagocytosis can be assessed by PMN as described in Example 14.

The term “complement-dependent cellular cytotoxicity” (“CDCC”) as used herein expresses a complement receptor that recognizes a complement 3 (C3) cleavage product covalently bound to a target cell or virion as a result of antibody-mediated complement activation. It is intended to refer to the mechanism of killing of a target cell or virion by a cell. CDCC can be evaluated in a similar manner as described for ADCC.

As used herein, the term “plasma half-life” refers to the time it takes to reduce the concentration of a polypeptide in blood plasma to half its initial concentration during removal (after the distribution phase). In the case of antibodies, the distribution phase will typically be 1-3 days, during which there is about a 50% reduction in blood plasma concentration due to redistribution between plasma and tissue. Plasma half-life can be measured by methods well known in the art.

The term “plasma clearance rate” as used herein is a quantitative measure of the rate at which a polypeptide is removed from the blood upon administration to a living organism. The plasma clearance rate can be calculated as dose/AUC (mL/day/kg), where the AUC value (area under the curve) is determined from the concentration-time curve according to Example 37.

As used herein, the term “downmodulation” is intended to refer to a process of reducing the number of molecules, such as antibodies or receptors, on a cell surface, eg by binding of an antibody to a receptor.

As used herein, the term “internalization” is intended to refer to any mechanism by which an antibody or Fc-containing polypeptide is internalized from the cell-surface and/or from the surrounding medium into the target-expressing cell, eg, through endocytosis. Internalization of an antibody can be assessed using a direct assay that measures the amount of internalized antibody (e.g., the lysosome colocalization assay described in Example 26).

As used herein, the term “antibody-drug conjugate” refers to an antibody or Fc-containing polypeptide having specificity for at least one type of malignant cell, a drug, and a linker that couples the drug, eg, to an antibody. The linker is cleavable or non-cleavable in the presence of malignant cells; Here the antibody-drug conjugate kills malignant cells.

The term "antibody-drug conjugate uptake" as used herein refers to the process by which an antibody-drug conjugate binds to a target on a cell and then is taken up/predated by the cell membrane and introduced into a cell. Antibody-drug conjugate uptake can be assessed as "antibody-mediated internalization and cell death by anti-TF ADC in an in vitro killing assay" as described in WO 2011/157741.

As used herein, the term “apoptosis” refers to the process of programmed cell death (PCD) that can occur in a cell. Biochemical events lead to characteristic cellular changes (morphology) and death. These changes include bubble formation, cell contraction, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. Binding of antibodies to specific receptors can induce apoptosis.

Fc-receptor binding can be measured indirectly as described in Example 12.

The term “FcRn” as used herein is intended to refer to the neonatal Fc receptor, which is an Fc receptor. It was first discovered in rodents as a unique receptor capable of transporting IgG from maternal milk to the neonatal bloodstream across the intestinal epithelium of rodent neonates. Further studies have revealed similar receptors in humans. However, in the human placenta, it has been found to help facilitate transport of maternal IgG to the growing fetus, and has also been found to play a role in the monitoring of IgG turnover. FcRn binds IgG at an acidic pH of 6.0-6.5, but not at neutral or higher pH. Thus, FcRn is able to bind IgG from the intestinal cavity (inside of the intestine) at a weakly acidic pH and ensures efficient one-way transport to the basolateral flank (inside the body) where the pH is neutral to basic (pH 7.0-7.5). This receptor also plays a role in the adult salvage of IgG through its occurrence in the pathway of endothelial cells in endothelial cells. The FcRn receptor in the acidic endosome binds to the internalized IgG through negative cell action, recycles it to the cell surface and releases it at the basic pH of the blood, preventing its lysosomal degradation. This mechanism may provide an explanation for the greater half-life of IgG in blood compared to other isotypes. Examples 13 and 34 describe assays showing IgG binding to FcRn at pH 6.0 in ELISA.

The term “protein A” as used herein is intended to refer to a 56 kDa MSCRAMM surface protein first found in the cell wall of the bacterium Staphylococcus aureus. It is encoded by the spa gene, and its regulation is controlled by DNA topology, cellular osmolality, and a two-complement system called ArlS-ArlR. It is useful in biochemical studies because of its ability to bind immunoglobulins. It consists of five homologous Ig-binding domains that fold into triple-helix bundles. Each domain is capable of binding proteins from many mammalian species, most particularly IgG. It binds to the heavy chain Fc region of most immunoglobulins (overlapping with the conserved binding site of the FcRn receptor) and also interacts with the Fab region of the human VH3 family. Through this interaction in the serum, the IgG molecule binds to the bacterium through its Fc region instead of through its Fab region where the bacterium destroys opsonization, complement activation and phagocytosis.

The term “protein G” is intended to refer to an immunoglobulin-binding protein expressed in Group C and G Streptococcus bacteria that is similar to Protein A but has different specificities. It is a 65-kDa (G148 protein G) and 58 kDa (C40 protein G) cell surface protein useful for purifying antibodies through their binding to the Fc region.

How to affect the CDC of the polypeptide

All embodiments described herein with reference to the parent antibody, first parent antibody or second parent antibody also apply to other parental, first parental or second parental polypeptides comprising the Fc-domain and binding region of an immunoglobulin. It should be understood as possible.

In one aspect, the present invention provides a parent polypeptide comprising an Fc domain and a binding region of an immunoglobulin at one or more amino acid residue(s) selected from the group corresponding to E430X, E345X, and S440W in the Fc region of a human IgG1 heavy chain. It relates to a method of increasing the complement-dependent cytotoxicity (CDC) of said parental polypeptide comprising introducing a mutation.

In one embodiment, the parental polypeptide may be a parental antibody comprising an Fc domain and an antigen-binding region of an immunoglobulin.

Introducing a mutation in a parent polypeptide according to the methods or uses of the present invention results in a variant polypeptide (also referred to herein as a “variant”). Thus, the method(s) of the invention can be carried out such that any variant or variant polypeptide as described herein is obtained.

The variant polypeptide obtained from the method or use of the invention has an increased CDC compared to the parent polypeptide. Typically, the effect of a polypeptide on effector function can be determined by the EC ₅₀ value, which is the concentration of the polypeptide required to obtain half the maximum lysis value.

Maximum dissolution is the dissolution obtained when a saturated amount of the polypeptide is used, where saturation is intended to refer to the amount of polypeptide to which all targets to the polypeptide are bound by the polypeptide.

The terms "increased CDC", "improved CDC", "increased effector function" or "improved effector function" in the context of the present invention refers to the presence of a decrease in the EC ₅₀ value of a variant polypeptide compared to the parent polypeptide. do. The decrease in the EC ₅₀ value can be, for example, at least or about 2 times, such as at least or about 3 times, or at least or about 5 times, or at least or about 10 times. Alternatively, "increased CDC", "improved CDC", "increased effector function" or "improved effector function" means that the parental polypeptide lyses less than 100% of all cells. The maximum amount (if the total amount of cells is set to 100%) is, for example, 10% to 100% of all cells, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60 %, about 70%, about 80%, about 90%, and about 100%.

Variants are increased or improved by cloning the variable domains of the IgG1-005 or IgG1-7D8 heavy chain as variants and testing their efficacy in a CDC assay as described for example for Daudi (Example 6) and Wien (Example 10). Can be tested for effector function. Using IgG1-7D8 HC variable domains and Daudi cells, the increase is an EC ₅₀ (concentration at which 1/2 of the maximal lysis is observed), such as about 2 times lower than the EC ₅₀ of IgG1-7D8 under the conditions studied. It will be defined by an EC ₅₀ value that is lower by a factor of, about 3, about 5, about 10 or more than 10 times. Using the IgG1-005 HC variable domain and Daudi cells, the increase is an EC ₅₀ (concentration at which 1/2 of the maximal lysis is observed), e.g., about 2 times lower than the EC ₅₀ of IgG1-005 under the conditions studied. It will be defined by an EC ₅₀ value that is lower by a factor of, about 3, about 5, about 10 or more than 10 times. Using the IgG1-7D8 HC variable domain and Wien133 cells, the increase is an EC ₅₀ (concentration at which 1/2 of the maximum lysis is observed), such as about 2 times lower than the EC ₅₀ of IgG1-7D8 under the conditions studied. It will be defined by an EC ₅₀ value that is lower by a factor of, about 3, about 5, about 10 or more than 10 times. Using IgG1-005 HC variable domain and Wien133 cells, the increase ranges from 10% to 100% of all cells, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, It will be defined as an increase in maximum dissolution by about 70%, about 80%, about 90%, and about 100%. In addition, an increase in the CDC potency EC ₅₀ is lower by more than twice the EC ₅₀ of IgG1-005 under the experimental conditions (that is 1/2 of the maximum observed in the melting possible dissolution of the Wien133 cell concentration detection condition), such as about It can be defined by a lower EC ₅₀ value by 2 times, about 3 times, about 5 times, about 10 times or more than 10 times.

The inventors of the present invention have surprisingly found that mutations at this specific position are directed against the CDC of variant antibodies obtained by introducing one or more mutation(s) into the parent antibody according to the method of the invention (e.g., as shown in Example 19). It was found to show an improved effect. Without being bound by theory, it is believed that oligomerization is stimulated by substituting one or more amino acid(s) from the group of positions mentioned above. The antibody binds with greater avidity (exemplified in Example 2; direct labeling of IgG-7D8-E345R increases binding to Daudi cells compared to IgG-7D8-WT), so that the antibody binds to cells for a longer time And thereby different effector functions such as increased C1q binding, C1q potency CDC, ADCC, internalization, ADCP, and/or in vivo potency. These effects are described in Examples 4 (C1q binding on cells), Example 5 (C1q efficacy in CDC assay), Examples 6, 7, 27, 28, 29, and 35 (CDC assay), Example 12 (ADCC) , Example 26 (internalization), Examples 21 and 22 (in vivo Efficacy), plasma clearance rate (Example 37), FcRn binding (Example 34), and target-independent fluid phase complement activation (Example 36).

Thus, mutations in amino acid residues selected from those corresponding to E430X, such as E430G, E430S, E430F, or E430T, E345X, such as E345K, E345Q, E345R, or E345Y, S440Y and S440W in the Fc region of a human IgG1 heavy chain are also present It may be referred to as a "single mutant" aspect or "CDC-enhancing mutation" in the context of the invention.

Thus, in one embodiment, the mutation in one or more amino acid residue(s) in the method of increasing CDC is at E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, and S440W in the Fc region of a human IgGl heavy chain. It is selected from the corresponding group.

In a preferred embodiment, the mutation in one or more amino acid residue(s) in the method of increasing CDC is selected from the group corresponding to E430G, E430S, E345K, and E345Q in the Fc region of a human IgG1 heavy chain.

In one embodiment, the parental polypeptide is a parental antibody comprising an Fc domain and an antigen-binding region of an immunoglobulin.

In another aspect, the invention also provides a parental polypeptide comprising an Fc domain and a binding region of an immunoglobulin, in the Fc region of a human IgG1 heavy chain, a mutation at one or more amino acid residue(s) corresponding to E430X, E345X, and S440W. And wherein X is any amino acid, such as a naturally occurring amino acid, to a method of increasing CDC and antibody dependent cell-mediated cytotoxicity (ADCC) of said parental polypeptide.

In one embodiment, the mutation in one or more amino acid residue(s) is selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, and S440W in the Fc region of a human IgG1 heavy chain.

In a preferred embodiment, the mutation in one or more amino acid residue(s) is selected from the group corresponding to positions E345R, E430T, and E430F in the Fc region of a human IgG1 heavy chain.

In one embodiment, at least one other effector function of the antibody, such as Clq-binding, complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC), Fc-gamma receptor-binding, protein A-binding, protein G-binding , ADCP, complement-dependent cellular cytotoxicity (CDCC), complement-promoting cytotoxicity, antibody mediated binding of opsonized antibodies to complement receptors, antibody mediated phagocytosis (ADCP), internalization, apoptosis, and/or The binding of opsonized antibodies to complement receptors, such as ADCC, is also increased.

In one embodiment, the parental polypeptide is a parental antibody comprising an Fc domain and an antigen-binding region of an immunoglobulin.

In one embodiment, the CDC of the parent antibody increases when the parent antibody binds to its antigen on an antigen-expressing cell, cell membrane, or virion.

In one embodiment, the parent antibody is a monospecific, bispecific, or multispecific antibody.

In a further aspect, the present invention provides a mutation in one or more amino acid residue(s) selected from the group corresponding to E430X, E345X, S440Y and S440W in the Fc region of a human IgG1 heavy chain to the first and/or second CH2-CH3 region. Includes introducing,

Wherein the first CH2-CH3 region comprises an additional amino acid mutation at a position selected from positions corresponding to K409, T366, L368, K370, D399, F405, and Y407 in the Fc region of a human IgG1 heavy chain; The second CH2-CH3 region comprises an additional amino acid mutation at a position selected from positions corresponding to F405, T366, L368, K370, D399, Y407, and K409 in the Fc region of a human IgG1 heavy chain, and the first CH2-CH3 region A first polypeptide comprising a first CH2-CH3 region and a first antigen-binding region of an immunoglobulin, wherein the additional amino acid mutation in is different from the additional amino acid mutation in the second CH2-CH3 region, and A bispecific antibody comprising a second polypeptide comprising a second CH2-CH3 region of a munoglobulin and a second antigen-binding region, wherein the first and second antigen-binding regions are the same antigen or different epitopes on different antigens. Binds to) of the parent antibody, which is a method of increasing the complement-dependent cytotoxicity (CDC).

In one embodiment, the mutation in one or more amino acid residue(s) is selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W in the Fc region of a human IgG1 heavy chain. .

In a preferred embodiment, the mutation at one or more amino acid residue(s) is selected from the group corresponding to E430G, E430S, E345K, and E345Q in the Fc region of a human IgG1 heavy chain.

In one embodiment, the method comprises introducing a mutation in only one of the first or second polypeptides of the bispecific antibody.

In one embodiment, the method comprises introducing a mutation in both the first and second polypeptides of the bispecific antibody.

In a preferred embodiment, the additional amino acid mutation in the first CH2-CH3 region is at a position corresponding to K409 in the Fc region of a human IgG1 heavy chain (eg, K409R); A further amino acid mutation in the second CH2-CH3 region is at a position corresponding to F405 in the Fc region of a human IgG1 heavy chain (eg, F405L).

The present inventors also found that the introduction of a mutation at an amino acid residue corresponding to K439 or S440 in the Fc region of a human IgG1 heavy chain into the parent antibody reduces the effector function of the parent antibody (Examples 5, 6 and 10).

As shown in Example 6, the amino acid substitution at position K439E or S440K as a “single-mutant” reduced CDC compared to either of the first mutations according to the method of the present invention.

The variant antibody obtained by this method of reducing effector function has a reduced effector function compared to the parent antibody. Typically, the effect of an antibody on effector function can be measured by the EC ₅₀ value, which is the concentration of antibody required to obtain half the maximum lysis value.

Maximum lysis is the lysis obtained when a saturating amount of antibody is used, where saturation is intended to refer to the amount of antibody to which all antigens to the antibody are bound by the antibody.

The term “reduction in effector function” refers in the context of the present invention to the presence of an increase in the EC ₅₀ value of a variant antibody compared to the parent antibody. The increase in the EC ₅₀ value can be, for example, at least or about 2 times, such as at least or about 3 times, or at least or about 5 times, or at least or about 10 times. Alternatively, "reduction in effector function" means that under conditions in which the parent antibody lyses less than 100% of all cells, the maximum amount of lysed cells, for example 10% to 100%, such as about 10% of all cells, By about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100%.

Variants are reduced effectors by cloning the variable domains of the IgG1-005 or IgG1-7D8 heavy chains as variants and testing their efficacy in a CDC assay as described for example for Daudi cells (Example 6) and Wien133 cells (Example 10). Can be tested for function. Using the IgG1-7D8 HC variable domain and Daudi cells, the reduction is an EC ₅₀ (concentration at which 1/2 of the maximal lysis is observed), such as about 2 times lower than the EC ₅₀ of IgG1-7D8 under the conditions studied. It will be defined by an EC ₅₀ value that is lower by a factor of, about 3, about 5, about 10 or more than 10 times. Using the IgG1-005 HC variable domain and Daudi cells, the reduction is an EC ₅₀ (concentration at which 1/2 of the maximal lysis is observed), which is about 2 times lower than the EC ₅₀ of IgG1-005 under the conditions studied. It will be defined by an EC ₅₀ value that is lower by a factor of, about 3, about 5, about 10 or more than 10 times. Using the IgG1-7D8 HC variable domain and Wien133 cells, the reduction is an EC ₅₀ (concentration at which 1/2 of the maximal lysis is observed), such as about 2 times lower than the EC ₅₀ of IgG1-7D8 under the conditions studied. It will be defined by an EC ₅₀ value that is lower by a factor of, about 3, about 5, about 10 or more than 10 times. Using the IgG1-005 HC variable domain and Wien133 cells, the reduction is from 10% to 100% of all cells, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about It will be defined as a reduction in maximum dissolution of 70%, about 80%, about 90%, and about 100%. In addition, the decrease in CDC efficacy is an EC ₅₀ (concentration at which lysis of Wien133 cells is observed 1/2 of the maximum lysis under detectable conditions), which is less than 2 times lower than the EC ₅₀ of IgG1-005 under the conditions studied. It can be defined by a lower EC ₅₀ value by 2 times, about 3 times, about 5 times, about 10 times or more than 10 times.

In a further aspect, the invention relates to a method according to the invention and to a method according to embodiments disclosed herein, which method introduces a mutation at one or more positions other than S440Y and S440W, and

(i) a mutation in each of the amino acid residues corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain (however, the mutation in S440 is not S440Y or S440W),

(ii) mutations in each of the amino acid residues corresponding to K447 and 448 in the Fc region of a human IgG1 heavy chain, such as K447K/R/H and 448E/D in the Fc region of a human IgG1 heavy chain, preferably the Fc region of a human IgG1 heavy chain K447K and 448E in, or,

(iii) mutations in each of the amino acid residues corresponding to K447, 448 and 449 in the Fc region of a human IgG1 heavy chain, such as K447D/E, 448K/R/H and 449P in the Fc region of a human IgG1 heavy chain, preferably human IgG1 K447E, 448K and 449P in the Fc region of the heavy chain

Including the additional introduction of.

With respect to the embodiment in which additional mutations are introduced as described in step (ii) or (iii) above, it should be noted that under normal circumstances the lysine at position K447 is removed during antibody production in the cell. This can be prevented by protecting position K447 by adding one or more additional amino acid residues (eg, 448 or 448/449). It is also described in WO 2013/004841 (Genmab A/S).

In one embodiment, the method comprises introducing a mutation at one or more positions other than S440Y and S440W, and further introducing a mutation at each of the amino acid residues corresponding to K439 and/or S440 in the Fc region of a human IgG1 heavy chain. It includes, provided that the mutation in S440 is not S440Y or S440W.

In a preferred embodiment, the mutation at the position corresponding to K439 in the Fc region of a human IgG1 heavy chain is K439D/E, and/or the mutation at the position corresponding to S440 in the Fc region of a human IgG1 heavy chain is S440K/R.

In one embodiment, the parental polypeptide is a parental antibody comprising an Fc domain and an antigen-binding region of an immunoglobulin.

In one embodiment, the parent antibody is a monospecific, bispecific, or multispecific antibody. Bispecific antibodies can be any of the embodiments described herein.

Any of the mutations listed in Table 1 can be introduced into the bispecific antibody. Example 24 shows that introduction of the E345R mutation into a bispecific CD20xEGFR antibody enhances CDC efficacy. Examples 23, 29 and 30 also describe some of the different bispecific antibodies comprising the mutations according to the invention.

The introduction of mutations at both amino acid residues corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain in the parent antibody (provided that the mutation at S440 is not S440Y or S440W) is also referred to herein as a "double mutant" aspect. Is referred to. The S440Y and S440W mutations have been found to increase CDC when introduced into the parental polypeptide as described elsewhere.

As also described elsewhere, the inventors of the present invention have found that introducing an identified mutation in the amino acid residue corresponding to either K439 or S440 in the Fc region of a human IgG1 heavy chain reduces effector function ( Examples 5, 6, 10). However, when inhibitory mutations at both amino acid residues corresponding to K439 and S440 in the Fc region of the human IgG1 heavy chain are introduced, the decrease in effector function is restored, whereby the effector function is free of mutations in the K439 and S440 mutations. It becomes similar to the effector function of the parent antibody. However, without being bound by any theory, it is believed that the presence of the K439 and S440 mutations limits the induction of effector function to an oligomeric complex consisting only of antibodies comprising both the K439 and S440 mutations. Thus, if the K439 and S440 mutations are included in the therapeutic antibody, it is not limited to any theory, and when such therapeutic antibodies are administered to a patient, the induction of effector function contains therapeutic antibodies comprising the K439/S440 mutations, but K439 and It is believed that the patient's own antibody not containing the S440 mutation is limited to an oligomeric antibody complex that does not contain, thereby limiting any potential side effects caused by the interaction of the therapeutic antibody with the patient's own antibody.

When the mutation at position K439 and/or S440 is combined with the first mutation, an enhancement of CDC is obtained and the specificity of CDC is increased. In a similar manner, enhancement of CDC and its increased specificity can be obtained by introducing the mutations disclosed in embodiments (ii) and (iii) above.

In another aspect, the present invention provides a mutation in one or more amino acid residue(s) selected from the group corresponding to E430X, E345X, S440Y, and S440W in the Fc region of a human IgG1 heavy chain at least the first and/or second parental polypeptide. Wherein at least the first and second parental polypeptides each comprise an Fc domain and a binding region of an immunoglobulin, wherein the complement-dependent cytotoxicity of a combination of at least first and second parental polypeptides ( CDC).

In one embodiment, the method eliminates at least a mutation in one or more amino acid residues selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W in the Fc region of a human IgG1 heavy chain. And/or the second parental polypeptide.

In a preferred embodiment, the method comprises a mutation in at least one of the first and/or second parental polypeptides selected from the group corresponding to E430G, E430S, E345K, and E345Q in the Fc region of a human IgG1 heavy chain. Includes introducing.

In one embodiment, the method comprises introducing a mutation, which may be the same or different, into both the first and second parental polypeptides.

In a further embodiment, the method

(i) Introducing a mutation in one or more amino acid residues selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W in the Fc region of a human IgG1 heavy chain into the first parent polypeptide Doing,

(ii) a second that does not contain a mutation in one or more amino acid residues selected from the group corresponding to mutations E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W in the Fc region of a human IgG1 heavy chain Includes providing the parent polypeptide.

In one embodiment, the method comprises introducing into the first parent polypeptide a mutation in one or more amino acid residue(s) selected from the group corresponding to E430G, E430S, E345K, or E345Q in the Fc region of a human IgG1 heavy chain.

In a further embodiment, the mutation at one or more positions is other than S440Y and S440W, wherein the method is

(i) introducing into the first parental polypeptide a second mutation at the amino acid residue corresponding to position K439 in the Fc region of a human IgG1 heavy chain; And

(ii) introducing a second mutation in the amino acid residue corresponding to S440 in the Fc region of the human IgG1 heavy chain to the second parental polypeptide, provided that the mutation is not S440Y or S440W, the step (i ) And (ii) alternatively

(iii) introducing a second mutation at the amino acid residue corresponding to position S440 in the Fc region of the human IgG1 heavy chain, provided that the mutation is not S440Y or S440W;

(iv) It may be a step of introducing a second mutation at an amino acid residue corresponding to position K439 in the Fc region of a human IgG1 heavy chain into the second parental polypeptide.

The second parental polypeptide can itself be any parental polypeptide that does not provide a sufficient CDC response upon binding to the target cell.

Thus, without being bound by theory, it provides a first variant polypeptide comprising a mutation in one or more amino acid residue(s) according to the above list (therefore the variant polypeptide has an increased CDC response), and such mutation(s) A method of providing a second variant polypeptide that does not contain (thereby obtaining a CDC response of the second parent polypeptide) is contemplated.

A method of combining a first antibody comprising one of the above mutations capable of increasing CDC as shown in Example 31 with a second antibody not modified according to the present invention increases the CDC of the combination. Thus, in one embodiment, such a method is used to combine a therapeutic antibody as a second antibody that has proven safe but is not sufficiently efficient (or where increased efficiency is desired) with a first antibody comprising a mutation to produce an efficacious combination. It is believed to be available for use.

Examples of suitable second antibodies that do not contain mutations in amino acid residues selected from those corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W in the Fc region of a human IgG1 heavy chain are: Including, but not limited to, any of; (90Y) clebatuzumab tetraxetane; (90Y) tacatuzumab tetraxetane; (99mTc) panolesomab; (99mTc) nofetumomab merpentan; (99mTc) pintumomab; 3F8; 8H9; Abagobomab; Avatarcept; Absiksimab; Actoxumab; Adalimumab; Adecatumumab; Apelimoab; Aflibercept; Afutuzumab; Alachizumab pegol; Albiglutide; ALD518; Allefacept; Alemtuzumab; Alirocumab; Altumomab; Altumomab pentate; Albircept sudotox; Amatuximab; AMG714/HuMax-IL15; Anatumomab Mefenatox; Anleukinzumab (=IMA-638); Apolizumab; Axitumomab; Acelizumab; Atalcicept; Atinumab; Atlizumab (= tocilizumab); Atorolimumab; Baminercept; Vafinuzumab; Basiliximab; Babituximab; Bektumomab; Belatacept; Belimumab; Benralizumab; Bertilimumab; Becilesomab; Bevacizumab; Bezlotoxumab; Beechromab; Biparcept; Vibatuzumab; Vibatuzumab mertansine; Blinatumomab; Blosozumab; Brentuximab vedotin; Briakinumab; Briovacept; Brodalumab; Kanakinumab; Cantuzumab mertansine; Cantuzumab ravtansine; Caplatizumab; Capromab; Capromap pendetide; Karlumab; Catumoxomab; CC49; Sedelizumab; Sertolizumab pegol; Cetuximab; Ch.14.18; Sitatuzumab bogatox; Drinking water tumumab; Clazakizumab; Clenoliximab; Clebatuzumab tetraxetane; Conatumumab; Concept; CR6261; Crenezumab; Dasetuzumab; Daclizumab; Dalantercept; Dalotuzumab; Daratumumab; Dempizumab; Denosumab; Detumomab; Dorlimomab Aritox; Drostumab; Dullaglutide; Ecromeximab; Eculizumab; Edobacomab; Edrecolomab; Efalizumab; Efunumab; Elotuzumab; Elcilimomab; Enabatuzumab; Enrimomab; Enrimomab Pegol; Enokizumab; Ensituximab; Epitumomab; Epitumomab situxetan; Epratuzumab; Erlizumab; Ertumaxomab; Etanercept; Etaracizumab; Etrolizumab; Exbirumab; Panolesomab; Paralimomab; Parletuzumab; Pasinumab; FBTA05; Felbizumab; Pezakinumab; Piclatuzumab; Pigitumumab; Flanvolumab; Pontolizumab; Foralumab; Poravirumab; Presolimumab; Fulanumab; Galiximab; Ganitumab; Gantenerumab; Gabilimomab; Gemtuzumab; Gemtuzumab ozogamicin; Gebokizumab; Girentuximab; Glembatumumab; Glembatumumab vedotin; Golimumab; Gomilliximab; GS6624; Anti-CD74 antibody; Anti-cMet antibodies as disclosed in WO 2011/110642; Anti-Her2 antibodies as disclosed in WO 2011/147986 or WO 2011/147982; Anti-IL8 antibodies as disclosed in WO 2004/058797; Anti-TAC antibodies as disclosed in WO 2004/045512; Anti-tissue factor (TF) antibodies as disclosed in WO 2010/066803 or WO 2011/157741; Ibalizumab; Ibritumomab Tiuxetan; Icrucumab; Igobomab; Temporarily; Inclacumab; Indatuximab labtansine; Infliximab; Inolimomab; Inotuzumab ozogamicin; Intetumumab; Iodine (124I) girentuximab; Ipilimumab; Iratumumab; Itolizumab; Ixekizumab; Keliximab; Rabetuzumab; Lebrikizumab; Remalesomab; Renercept; Lerdelimumab; Lexatumumab; Ribirumab; Lintuzumab; Lorbotuzumab mertansine; Lucatumumab; Lumiliximab; Mapatumumab; Maslimomab; Matuzumab; Mabrilimumab; Mepolizumab; Metelimumab; Milatuzumab; Minretumomab; Mirococept; Mitumomab; Mogamulizumab; Morolimumab; Motabizumab; Moksetumomab; Pasudotox; Muromonap-CD3; Nacolomab tapenatox; Namilumab; Naftumomab estafenatox; Narnatumab; Natalizumab; Nebacumab; Nesitumumab; Nerelimomab; Nimotuzumab; Nivolumab; Nofetumomab; Merpentane; Obinutuzumab; Okaratuzumab; Ocrelizumab; Odulimomab; Ofatumumab; Olatumab; Olokizumab; Omalizumab; Onartuzumab; Onercept; Oportuzumab Monatox; Oregobomab; Otelixizumab; Oxelumab; Ozoralizumab; Pagibaximab; Palivizumab; Panitumumab; Panobacumab; Pascolizumab; Pateklizumab; Patritumab; Pegsunercept; Femtumomab; Pertuzumab; Pexelizumab; Pintumomab; Paclaculumab; Phonezumab; Priliximab; Pritumumab; PRO 140; Quilizumab; Lacotumomab; Radretumab; Rapivirumab; Ramuchirumab; Ranibizumab; Laxibacumab; Regavirumab; Reslizumab; RG1507/HuMax-IGF1R; RG1512/HuMax-p selectin; Lilonacept; Rilotumumab; Rituximab; Lovatumumab; Rolledumab; Lomosozumab; Rontalizumab; Loblizumab; Ruflizumab; Samalizumab; Sarilumab; Satumomab; Satumomab pendetide; Secukinumab; Sebirumab; Sibrotuzumab; Sifalimumab; Siltuximab; Siplizumab; Sirukumab; Solanezumab; Solitomab; Sonebzizumab; Handtuzumab; Sotatercept; Stamulumab; Sulesomab; Subizumab; Tabalumab; Tacatuzumab tetraxetane; Tadochizumab; Talizumab; Tanezumab; Taplitumomab Poptox; Tefibazumab; Telemomab aritox; Tenatumomab; Teneliximab; Teflizumab; Teprotumumab; TGN1412; Ticilimumab (= tremelimumab); Tigatuzumab; TNX-650; Tocilizumab (= atlizumab); Toralizumab; Torabcel; Tositumomab; Tralokinumab; Trastuzumab; Trastuzumab emtansine; TRBS07; Trevanaib; Tregalizumab; Tremelimumab; Tucotuzumab cellmorukin; Tuvirumab; Ublituximab; Urelumab; Urtoxazumab; Ustekinumab; Bapaliximab; Batellizumab; Vedolizumab; Beltuzumab; Bepalimomab; Besencumab; Vicilizumab; Volosicimab; Borsetuzumab mapodotin; Botumumab; Zalutumumab; Zanolimumab; Ziralimumab; And Jolimomab Aritox.

The first and second variant antibodies will favor oligomerization with each other compared to any wild type or naturally occurring antibody as shown in Example 10.

In one embodiment, the mutation at the position corresponding to K439 in the Fc region of a human IgG1 heavy chain is K439D/E and/or the mutation at the position corresponding to S440 in the Fc region of a human IgG1 heavy chain is S440K/R.

As such, an increase in specificity is associated with "induction of CDC". Thus, the method is, in one embodiment, a method of increasing the specificity of induction of effector function by a combination of at least a first and a second parent polypeptide.

By performing a method of increasing the specificity of or induction of effector function by at least the combination of the first and second parental polypeptides, a combination of a first variant and a second variant polypeptide is obtained.

By introducing a mutation in K439 or S440 of the parental polypeptide, the variant polypeptide thus obtained has a reduced effector function compared to the parental polypeptide. However, as also described elsewhere herein, mutations in K439 and S440 may complement each other or restore the effector function of a polypeptide comprising both mutations. This ability of mutations in K439 and S440 to complement each other can be used similarly in the two polypeptides. Thus, the mutation in K439 is introduced into the first parental polypeptide and the mutation in S440 is introduced into the second parental polypeptide, or vice versa, the decrease in effector function is used in combination with the first and second variant polypeptides. As in the case, it is no longer observed. The terms "increased specificity" or "improved specificity" in this context means that the effector response induced by the combination of a first variant polypeptide comprising a mutation at K439 and a second variant polypeptide comprising a mutation at S440 is a mutation at K439. Is higher than the effector response induced by the first variant polypeptide comprising or by the second variant polypeptide comprising a mutation in S440.

The specificity of oligomerization is increased by the introduction of both amino acid substitutions at K439 and S440.

When combining the mutation at position K439 and/or S440 with the first mutation, CDC is enhanced and the specificity of CDC is increased.

In one embodiment, at least the first and second parental polypeptides bind to the same binding site, or to the same epitope with respect to the antibody.

In one embodiment, at least the first and second parental polypeptides bind to different binding sites on the same target, or, with respect to the antibody, to different epitopes on the same antigen.

In one embodiment, at least the first and second parental polypeptides bind different epitopes on different targets.

In one embodiment, the first and second parental polypeptides are first and second parental antibodies having the same or different VL and VH sequences.

In one embodiment, the combination of at least a first and second parent polypeptide comprises one first parent polypeptide and one second polypeptide.

In one embodiment, specificity increases when the combination of the first and second parental polypeptide binds to an antigen or its binding site on an antigen-expressing cell, cell membrane, or virion.

Thus, in another aspect, the invention also provides for increasing the specificity of a polypeptide (e.g., of a CDC induced thereby) of a polypeptide when bound to its antigen on an antigen-expressing cell, cell membrane, or virion. Relates to the use of mutations on two or more amino acid residues, wherein

The first mutation is at the amino acid residue corresponding to K439 in the Fc region of a human IgG1 heavy chain;

The second mutation is at the amino acid residue corresponding to S440 in the Fc region of a human IgG1 heavy chain.

In one embodiment, the first and second parental polypeptides are first and second parental antibodies, respectively, comprising an Fc domain and an antigen-binding region of an immunoglobulin.

In one embodiment, the first and second parent antibodies are monospecific, bispecific or multispecific antibodies.

In one embodiment, the first and/or second parent antibody is a first polypeptide comprising a first CH2-CH3 region and a first antigen-binding region of an immunoglobulin, and a second CH2-CH3 region and a second antigen. -A bispecific antibody comprising a second polypeptide comprising a binding region, wherein the first and second antigen-binding regions bind to the same antigen or different epitopes on different antigens, and the first CH2-CH3 region is human An additional amino acid mutation at a position selected from positions corresponding to K409, T366, L368, K370, D399, F405, and Y407 in the Fc region of the IgG1 heavy chain; The second CH2-CH3 region comprises an additional amino acid mutation at a position selected from positions corresponding to F405, T366, L368, K370, D399, Y407, and K409 in the Fc region of a human IgG1 heavy chain, and the first CH2-CH3 region The further amino acid mutation at is different from the further amino acid mutation at the second CH2-CH3 region.

In a preferred embodiment, the first CH2-CH3 region comprises an additional amino acid mutation (eg, K409R) at a position corresponding to K409 in the Fc region of a human IgG1 heavy chain; The second CH2-CH3 region contains an additional amino acid mutation (eg, F405L) at a position corresponding to F405 in the Fc region of a human IgG1 heavy chain.

By performing this method, a combination of at least the first and second variant antibodies is obtained. At least the first and second variant antibodies obtained by this method, when combined, have an increased CDC compared to the combination of the first and second parent antibodies.

The term “increased CDC” is to be understood as described herein.

The first and/or second parent antibody can be any parent antibody as described herein.

The method of increasing the CDC of the combination of the first and second antibodies can be carried out so that a first and/or second variant antibody is obtained, in particular, having any of the properties of a variant antibody as described herein.

In one embodiment, at least the first and second parent antibodies bind the same epitope.

In one embodiment, at least the first and second parent antibodies bind different epitopes on the same antigen.

In one embodiment, at least the first and second parent antibodies bind different epitopes on different targets.

In one embodiment, the first and second parent antibodies have the same or different VL and VH sequences.

In one embodiment, the combination of at least first and second parent antibodies comprises one first parent antibody and one second antibody.

In one embodiment, the combination of at least the first and second parent antibodies comprises an additional parent antibody, such as a third, fourth or fifth parent antibody. In one embodiment, the first and second bispecific or multispecific parent antibodies are the same or different antibodies. In one embodiment, the first and second bispecific or multispecific parent antibodies bind different epitopes on the same or different antigens. Thus, in one embodiment, said at least first and second parent antibodies are bispecific or multispecific antibodies that bind to the same antigen or different epitopes on different antigens.

In one embodiment of the methods and/or uses of the invention, whether the parent antibody, the first parent antibody, or the second parent antibody, the parent antibody is a mutation other than the mutation of the invention that has been found to affect effector function. It may include. These other mutations may be introduced simultaneously or sequentially with the mutations of the present invention that affect effector function, but the methods or uses of the present invention are not limited to simultaneous or sequential introduction of mutations. The bispecific antibody may be any bispecific antibody, but the methods and uses of the present invention are not limited to any particular bispecific format as it is expected that different formats may be used.

In one embodiment, the method does not alter the parent polypeptide or antibody dependent cell-mediated cytotoxicity (ADCC) of the parent antibody.

In one embodiment, the method does not alter the binding of the parental polypeptide or parental antibody to the neonatal Fc receptor (FcRn) as determined by the method disclosed in Example 34.

In one embodiment, the method exceeds 30% binding of the parental polypeptide or parental antibody to the neonatal Fc receptor (FcRn) as determined by a change in absorbance at OD405 nm as determined by the method disclosed in Example 34, such as Do not increase or decrease by more than 20%, 10% or 5%.

In one embodiment, the method does not increase the apparent affinity of the parental polypeptide or parental antibody for the mouse neonatal Fc receptor (FcRn) by a factor of more than 0.5 or mouse FcRn as determined by the method disclosed in Example 34. Do not reduce the apparent affinity of the parent polypeptide or parent antibody for a fold by more than 2.

In one embodiment, the method does not alter the plasma clearance rate of the parental polypeptide or parental antibody as determined by the method disclosed in Example 37.

In one embodiment, the method increases the plasma clearance rate of the parent polypeptide or parent antibody by a fold greater than 3.0, such as a fold 2.5, fold 2.0, fold 1.5, or fold 1.2, as determined by the method disclosed in Example 37, or Or do not decrease.

In one embodiment, the method does not alter the target-independent fluid phase complement activation of the variant as determined by the method disclosed in Example 36.

In one embodiment, the method does not alter the plasma half-life of the parent polypeptide or parent antibody.

Any mutations described herein or combinations thereof can be introduced according to the methods of the present invention.

Mutations selected from exemplary or preferred amino acid substitutions are in suitable assays, such as those described in the Examples, that allow detection of oligomer formation and enhanced C1q-binding, complement activation, CDC, ADCC and/or internalization of the antigen-bound antibody. Can be tested. For example, C1q-avidity can be determined according to an assay similar to that described in Example 4 using cells expressing antigens for antibody variants. Exemplary CDC assays are shown in Examples 5, 6, 10, 16, 19, 22, 23, 24, 25, and 35. An exemplary ADCC assay is presented in Example 12. An exemplary internalization assay is presented in Example 26. Finally, to distinguish between mutations in amino acid residues directly involved in C1q-binding from mutations affecting oligomer formation, C1q-binding in the ELISA assay according to Example 3, for example Can be compared with C1q-binding in the cell-based assay according to 4, and plasma clearance rates can be compared according to the assay described in Example 37, FcRn binding comparison according to Example 34, and target-independent fluid phase complement activation It can be evaluated according to the assay in Example 36.

In one embodiment, mutations in one or more amino acid residue(s) may be amino acid substitutions, amino acid deletions or amino acid insertions.

In one embodiment, the mutation in one or more amino acid residue(s) is an amino acid deletion.

In one embodiment, the mutation in one or more amino acid residue(s) is an amino acid insertion.

In certain embodiments, the mutation in one or more amino acid residue(s) is an amino acid substitution.

In one embodiment, mutations in one or more amino acid residue(s) can be selected from any amino acid substitutions, amino acid deletions listed in Table 1.

Thus, in one embodiment, E345X is E345R, Q, N, K, Y, A, C, D, F, G, H, I, L, M, P, S, T, V, W, or Y; In particular E345A, D, G, H, K, N, Q, R, S, T, Y or W, or more particularly E345D, K, N, Q, R, or W; Or even more particularly E345R, Q, N, K, or Y. In a further preferred embodiment, E345X is E345K or E345Q.

In another further embodiment, E430X is E430T, S, G, F, H, A, C, D, I, K, L, M, N, P, Q, R, V, W, or Y; In particular, it may be E430T, S, G, F, or H. In a further preferred embodiment, E430X is E430G or E430S. In another embodiment, the mutation is optionally directly involved in C1q-binding as determined by comparing C1q-binding in an ELISA assay according to Example 3 with a C1q-binding in a cell-based assay according to Example 4. Not at the amino acid residue.

In one embodiment, the one or more mutation(s) is one mutation, ie no more than one mutation is introduced into the parent antibody.

In another embodiment, the method or use according to the invention comprises introducing a mutation in at least two of the amino acid residues of Table 1, such as 2, 3, 4, 5 or more.

Any combination of mutations described herein can be introduced according to the methods of the present invention.

In one embodiment, the method comprises in the parent polypeptide more than one mutation, such as 2, 3, 4, or 5, at an amino acid residue selected from the group corresponding to E345X, E430X, S440Y, and S440W in the Fc region of a human IgG1 heavy chain. Dogs, in particular 2 or 3 mutations. For example, at least one or more of the amino acid residues corresponding to E345X, E430X, S440Y, and S440W in the Fc region of a human IgG1 heavy chain may be mutated (e.g., two or both of E345X, E430X, S440Y, and S440W. , Optionally combined with mutations in one or more other amino acids listed in Table 1). The at least two mutations may be any amino acid residue substitution at position E430 or any amino acid residue substitution at position E345 in combination with S440Y or S440W, or any of position E430 in combination with any amino acid residue at position S440Y or S440W. It may be an amino acid substitution. In a further embodiment, 2 or 3 mutations in amino acid residues selected from the group corresponding to E430G, E430S, E345K, and E345Q in the Fc region of a human IgG1 heavy chain are introduced into the parent antibody.

This combination of two mutations in amino acid residues is selected from the group corresponding to E345X/E430X, E345X/S440Y, E345X/S440W, E430X/S440Y, and E430X/S440W in the Fc region of a human IgG1 heavy chain.

In the method or use according to the invention, CDC increases when the antibody binds its antigen.

Without being bound by any theory, CDC increases when the antibody binds its antigen, where the antigen is on an antigen-expressing cell, cell membrane or virion. In one embodiment, the Fc region of the IgG1 heavy chain comprises the sequence of residues 130-330 of SEQ ID NO:1.

The parent polypeptide or parent antibody may be any parent polypeptide or any parent antibody as described herein. Parental polypeptides and parental antibodies in this context are also intended to be first parental and second parental polypeptides and first parental and second parental antibodies.

In one embodiment, the parent antibody is a human IgG1, IgG2, IgG3 or IgG4, IgA1, IgA2, IgD, IgM or IgE antibody.

In one embodiment, the parent antibody is a human full length antibody, such as a human full length IgG1 antibody.

In one embodiment, the parent antibody, the first parent antibody and the second parent antibody are a human IgG1 antibody optionally comprising an Fc-region comprising SEQ ID NO: 1 or 5, e.g. IgG1m(za) or IgG1m(f) homologous It's a variant.

In one embodiment, the parent antibody is a human IgG2 antibody optionally comprising an Fc-region comprising SEQ ID NO: 2.

In one embodiment, the parent antibody is a human IgG3 antibody optionally comprising an Fc-region comprising SEQ ID NO: 3.

In one embodiment, the parent antibody is a human IgG4 antibody optionally comprising an Fc-region comprising SEQ ID NO: 4.

In one embodiment, the parent antibody is a bispecific antibody.

In one embodiment, the parent antibody is any antibody as described herein, e.g., an antibody fragment comprising at least a portion of an Fc-region, a monovalent antibody (WO2007059782 (described in Genmab); consisting of only two heavy chains). Heavy chain antibodies naturally occurring in, for example, camels (eg Hamers-Casterman (1993) Nature 363:446); ThioMab (Roche, WO2011069104), symmetrical and bispecific antibody-like molecules Phosphorus-strand-exchange engineered domain (SEED or seed-body) (Merck, WO2007110205); Triomab (Fregenius, Lindhofer et al. (1995 J Immunol 155:219)); FcΔAdp (regenerone, WO2010151792 ), azimetric scaffold (Zymeworks/Merck, WO2012/058768), mAb-Fv (Zencor, WO2011/028952), dual variable domain immunoglobulins (Abbot, DVD-Ig, U.S. Patent No. 7,612,181); dual Domain double head antibody (UniLever; Sanofi Aventis, WO20100226923), D-diabody (Imclon/Ely Lily), knob-into-hole antibody format (Genentech, WO9850431); Duobody (Genmab, WO 2011/131746); electrostatic Miracle Steering Antibody Format (Amgen, EP1870459 and WO 2009089004; Cheugai, US201000155133; Oncomed, WO2010129304A2); Bispecific IgG1 and IgG2 (Linat Neuroscience Corporation, WO11143545), CrossMAb (Roche, WO2011117329), LUZ- Y (Genentech), Biclonic (Merus), dual targeting domain antibody (GSK/Domantis), two-in-one antibody that recognizes two targets (Genentech, Novimune), cross-linked Mab (Carmanose Cancer Center), CovX-body (CovX/Pfizer), IgG-like bispecific (Imclones/Ely Lily, Shen, J., et al. J Immunol Met) hods, 2007. 318(1-2): p. 65-74]), and DIG-body and PIG-body (parmabusin), and dual-affinity retargeting molecules (Fc-DART or Ig-DART, macrogenix, WO/2008/157379, WO/2010/080538 ), an antibody containing an Fc-domain-like scFv-fusion, as well as an approach using a Zybadis (Zaingenia), a conventional light chain (Crusel/Merus, US7262028) or a conventional heavy chain (κλbody, Novimune) Fusion protein comprising a polypeptide sequence fused to a fragment (same as BsAb, Zimogenetics/BMS), Hercules (Biogen IDec) (US007951918), Scorpions (Imergent Biosolutions/Trubion), Ts2Ab (Medimum/AZ (Dimasi, N., et al. J Mol Biol, 2009. 393(3): p. 672-92), scFv fusion (Nopartis), scFv fusion (Zhangzhou Adam Biotech Inc. (CN 102250246), TvAb (Roche) (WO 2012025525, WO 2012025530), mAb ² (f-star) (WO2008/003116), and dual scFv-fusions. Further, unless otherwise specified, the term antibody is also a polyclonal antibody, monoclonal antibody. Raw antibodies (e.g., human monoclonal antibodies), e.g., antibody mixtures (recombinant polyclonal antibodies) produced by techniques used by Sympogen and Merus (oligoclonic), and antibody-like polypeptides, It should be understood to include, for example, chimeric antibodies and humanized antibodies The resulting antibodies can potentially have any isotype.

In another embodiment, the antigen is expressed on the surface of the cell.

In another embodiment, the cell is a human tumor cell.

In a further embodiment, the antigen is erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD38, CD138, CXCR5, c-Met, HERV-enveloped protein, periostin, Bigh3. , SPARC, BCR, CD79, CD37, EGFrvIII, IGFr, L1-CAM, AXL, tissue factor (TF), CD74, EpCAM and MRP3.

In another embodiment, the antigen is associated with a cell membrane.

In another embodiment, the antigen is associated with a virion, and optionally the antigen is included in the protein coat or lipid envelope of the virion.

In another embodiment, the antibody is a human antibody that optionally binds to at least one antigen selected from CD20 and CD38.

In another embodiment, the antibody binds to the same epitope as at least one of 7D8 and 005, and optionally comprises a variable heavy and/or variable light chain region of at least one of 7D8 and 005.

In any use according to the disclosed invention, the antibody without any mutations of the invention may be any parent antibody. Thus, use herein provides for any variant of such parent antibody.

In one embodiment, the effector function is Fc-receptor binding, including, for example, Fc-gamma receptor binding.

In one embodiment, the effector function is Fc-containing polypeptide internalization.

In one embodiment, the effector function is a combination of complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC).

The term "C1q-binding" as used herein, when used in connection with a variant or antibody of a parent antibody, is mediated by binding of a variant or antibody to a host tissue or factor, including various cells of the immune system (eg, effector cells). And any mechanism of the first component on the classical pathway of complement activation to become. C1q-binding of the antibody can be assessed using an ELISA (e.g., C1q-binding ELISA used in Examples 3 and 4), or the C1q efficacy can be evaluated in a CDC assay (e.g., in Example 5 CDC assay used). In a further embodiment, the C1q-binding ability of the antibody is determined according to the assay described in Example 4.

In all methods according to the disclosed invention, an antibody without any mutations of the invention may be any parent antibody. Thus, the methods herein provide for any variant of such parent antibody.

The parent antibody, first parent antibody, second parent antibody, or variant thereof obtained by the methods and/or uses of the invention can bind to any target as described herein.

Examples of targets or antigens to which the present invention can act are as follows; 5T4; ADAM-10; ADAM-12; ADAM17; AFP; AXL; ANGPT2 anthrax antigen; BSG; CAIX; CAXII; CA 72-4; Carcinoma Associated Antigen CTAA16.88; CCL11; CCL2; CCR4; CCR5; CCR6; CD2; CD3E; CD4; CD5; CD6; CD15; CD18; CD19; CD20; CD22; CD24; CD25; CD29; CD30; CD32B; CD33; CD37; CD38; CD40; CD40LG; CD44; CD47; CD52; CD56; CD66E; CD72; CD74; CD79a; CD79b; CD80; CD86; CD98; CD137; CD147; CD138; CD168; CD200; CD248; CD254; CD257; CDH3; CEA; CEACAM5; CEACAM6; CEACAM8; Claudin4; CS-1; CSF2RA; CSPG-4; CTLA4; Cripto; DLL4; ED-B; EFNA2; EGFR; Endothelin B receptor; ENPP3; EPCAM; ERBB2; ERBB3; FAP alpha; Fc gamma RI; FCER2; FGFR3; Fibrin II beta chain; FLT1; FOLH1; FOLR1; FRP-1; GD3 ganglioside; GDF2; GLP1R; Glypican-3; GPNMB; HBV (hepatitis B virus); HCMV (human cytomegalovirus); Heat shock protein 90 homologue [ Candida albicans ]; Herpes simplex virus gD glycoprotein; HGF; HIV-1; HIV-1 IIIB gp120 V3 loop; HLA-DRB (HLA-DR beta); Human respiratory syncytial virus, glycoprotein F; ICAM1; IFNA1; IFNA1; IFNB1 bispecific; IgE Fc; IGF1R; IGHE connection area; IL12B; IL13; IL15; IL17A; IL1A; IL1B; IL2RA; IL4; IL5; IL5RA; IL6; IL6R; IL9; Interleukin-2 receptor beta subunit; ITGA2; ITGA2B ITGB3; ITGA4 ITGB7; ITGA5; ITGAL; ITGAV_ITGB3; ITGB2; KDR; L1CAM; Lewis-y; Lipid A, domain of lipopolysaccharide LPS; LTA; MET; MMP14; MMp15; MST1R; MSTN; MUC1; MUC4; MUC16; MUC5AC; NCA-90 granulocyte cell antigen; Nectin 4; NGF; NRP; NY-ESO-1; OX40L; PLAC-1; PLGF; PDGFRA; PD1; PDL1; PSCA; Phosphatidylserine; PTK-7; Pseudomonas aeruginosa serotype IATS O11; RSV (human respiratory syncytial virus, glycoprotein F); ROR1; RTN4; SELL; SELP; STEAP1; Shiga-like toxin II B subunit [ Escherichia coli ]; SLAM7; SLC44A4; SOST; Staphylococcus epidermidis lipoteichoic acid; T cell receptor alpha_beta; TF; TGFB1; TGFB2; TMEFF2; TNC; TNF; TNFRSF10A; TNFRSF10B; TNFRSF12A; TNFSF13; TNFSF14; TNFSF2; TNFSF7; TRAILR2; TROP2; TYRP1; VAP-1; And vimentin.

In the main aspect, the present invention

(i) providing a mutated parent polypeptide or a combination of at least a first parent polypeptide and a second parent polypeptide according to any one of the embodiments disclosed herein; And

(ii) a preparation of the mutated parental polypeptide of step (i) or a mutated combination of at least a first parental polypeptide and a second parental polypeptide of step (i) is a cell, cell expressing the antigen in the presence of a human complement or effector cell. Contacting the membrane or virion

It relates to a method for inducing CDC against cells, cell membranes, or virions expressing a target to which the parent polypeptide comprising the Fc-domain and binding region of an immunoglobulin, including, to bind.

In one embodiment, any or all of the parent polypeptide, the first parent polypeptide and the second parent polypeptide may be antibodies.

In another embodiment, the method comprises ADCC, Fc-gamma receptor-binding, protein A-binding, protein G-binding, ADCP, complement-dependent cellular cytotoxicity (CDCC), complement-enhanced cytotoxicity, mediated by an antibody. Increases the binding of the opsonized antibody to the complement receptor, and additional effector function selected from any combination thereof.

In a further embodiment, the method also induces antibody-dependent cell-mediated cytotoxicity (ADCC).

In another further embodiment, the method also induces internalization of the Fc-containing polypeptide.

In one embodiment, the cells are human tumor cells or bacterial cells.

In another embodiment, the IgG1 parent antibody is a human IgG1 antibody.

In another embodiment, the first and second antigens are individually erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD25, CD32, CD37, CD38, CD74, CD138 , CXCR5, c-Met, HERV-enveloped protein, periostin, Bigh3, SPARC, BCR, CD79, EGFrvIII, IGFr, L1-CAM, AXL, tissue factor (TF), EpCAM and MRP3.

In another embodiment, the first and second parent antibodies are fully human antibodies, and optionally the first and second parent antibodies bind antigens individually selected from CD20 and CD38.

In a further embodiment, the first and second parent antibodies are individually selected from 7D8 and 005.

In a further embodiment, the cell is a bacterial cell.

In another embodiment, the bacterial cell is S. Aureus, S. Epidermidis , S. Pneumoniae (S. pneumonia), Bacillus anthraquinone system (Bacillus anthracis), Pseudomonas Ah Labor (Pseudomonas aeruginosa), chlamydia (Chlamydia) rugi on this. E. coli, Salmonella (Salmonella), Shigella (Shigella), Yersinia (Yersinia), S. Typhimurium ( S. typhimurium ), Neisseria meningitides ( Neisseria meningitides ) and Mycobacterium tuberculosis ( Mycobacterium tuberculosis ) is selected from the group consisting of .

In another embodiment, the first and/or second antigen is lipoteicosan (LTA) and optionally at least one of the first and second parent antibodies is pagibaximab.

In another embodiment, the antigen is expressed on a virion.

In another embodiment, the first and second antibodies bind the same antigen.

In another embodiment, the first and second antibodies comprise the same VH sequence, VL sequence, or both VH and VL sequences.

For the purposes of the present invention, the target cells expressing the antigen or otherwise associated with the antigen may be any prokaryotic or eukaryotic cell. Exemplary antigen-expressing cells include mammalian cells, particularly human cells, such as human cancer cells; And unicellular organisms such as bacteria, protozoa, and unicellular fungi such as yeast cells. Cell membranes that contain an antigen or otherwise associate with an antigen include partially and/or destroyed cell membranes derived from antigen-expressing cells. The antigen associated with the virion or viral particle may be included in the protein coat and/or lipid envelope of the virion or otherwise associated.

The target cell can be, for example, a human tumor cell. Suitable tumor antigens are any target or antigen as described herein, erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD25, CD32, CD37, CD38, CD74, CD138 , CXCR5, c-Met, HERV-envelope protein, periostin, Bigh3, SPARC, BCR, CD79, EGFrvIII, IGFR, L1-CAM, AXL, tissue factor (TF), EpCAM and MRP3. . Preferred antigens include CD20, CD38, HER2, EGFR, IGFR, CD25, CD74 and CD32. Exemplary antibodies are anti-CD20 antibody 7D8 as disclosed in WO 2004/035607, anti-CD38 antibody 005 as disclosed in WO 06/099875, anti-CD20 antibody 11B8 as disclosed in WO 2004/035607, WO 06/099875 Anti-CD38 antibody 003 as disclosed in, and anti-EGFr antibody 2F8 as disclosed in WO 02/100348. Examples of other specific antibodies are provided herein.

Alternatively, the target cell is a bacterial cell, such as, for example, S. Aureus, S. Epidermides, S. Pneumoniae, Bacillus anthracis, Pseudomonas aeruginosa, Chlamydia, E. Coli, Salmonella, Shigella, Yersinia, S. Typhimurium, Neisseria meningitides and Mycobacterium tuberculosis. Exemplary antigens include lipoteicosan (LTA), and exemplary antibodies include pagibaximab.

Alternatively, the target is a virus, fungal cell or other particle, such as, for example, West Nile virus, Dengue virus, Hepatitis C virus (HCV), human immunodeficiency virus (HIV), Human papillomavirus, Epstein-Barr virus, Herpesvirus, poxvirus, avian influenza virus, RVS, Aspergillus, Candida albicans, Cryptococcus And it may exist on the surface of histoplasma ( Histoplasma ).

In one embodiment, the contacting step (ii) occurs in vitro.

In one embodiment, the contacting step (ii) occurs in vivo.

In another embodiment, step (ii) comprises administering the variant to the subject.

In a further embodiment, the subject suffers from cancer, bacterial infection, or viral infection. The contacting step (ii) of the above-mentioned embodiments can take place in vitro or in vivo. In the latter case, step (ii) may further comprise administering the agent or agents to the subject, optionally the subject suffering from a cancer or bacterial infection. Further details for therapeutic use are provided below.

The first and second antibodies comprise antigen-binding regions capable of binding the same or different epitopes. These epitopes can be on the same or different targets.

In one embodiment, the first and second antibodies bind different epitopes on different targets. Such targets can be expressed on the same cell or cell type, or can be expressed on different cells or cell types. In such embodiments, the enhancement of effector function is induced only in cells or cell types expressing both targets, thereby reducing the risk of any concomitant damage to the cell or cell type that is not the cause of the disease being treated.

Without being bound by any theory, the enhancement of CDC is limited to target cells expressing two specific targets/antigens simultaneously as long as the first and second antibodies bind to the epitope found on the same cell, thereby It is believed that combined expression could be used to improve the selectivity of enhanced CDC induction.

Where the target is expressed on different cells or cell types, without being limited to theory, administration of any order of the first and second antibodies promotes CDC and also presumably of the second cell or cell type expressing the second target. It is believed that "mobilization" will improve other effector functions.

In one embodiment in which a combination of the first and second antibodies is used, step (ii) brings the cells simultaneously, individually, or sequentially with the mutated first and second parent antibodies in the presence of human complement and/or effector cells. It can be done by contacting with.

The present invention also provides a variant of the antibody comprising (i) a mutation in K439, which is K439E, and a mutation in S440, which is S440K or S440R in the Fc region of the antibody; And (ii) contacting the preparation of the variant with the cell in the presence of human complement and/or effector cells, to target cells, cell membranes, virions or other particles that associate with the antigen to which the IgG1 or IgG3 antibody binds. Methods for inducing a CDC or other effector response to, such as ADCC, are provided.

The present invention also comprises the steps of: (i) providing a first variant that is a first antibody comprising a K439E mutation and a second variant that is a second antibody comprising a S440K or S440R mutation; And (ii) contacting the cells with the preparations of the first and second variants simultaneously, separately or sequentially in the presence of human complement or effector cells, wherein the first antigen and the second antigen to which the first IgG1 antibody binds. Methods of inducing a CDC or other effector response, such as ADCC, against a target cell, cell membrane or virion that express a second antigen to which the antibody binds.

In individual and specific embodiments, the first and second antibodies are selected from (i) different antigens; (ii) different epitopes on the same antigen, (iii) the same epitope on the antigen, and (iv) the same epitope on the antigen and contain the same VH and/or VL sequences.

other way

In another major aspect, the present invention

(i) preparing at least one antibody comprising a mutation in one or more amino acid(s) selected from the group corresponding to E430X, E345X, S440Y and S440W in the Fc region of a human IgG1 heavy chain;

(ii) evaluating the C1q-activity of the antibody when bound to the surface of the antigen-expressing cell compared to the parent antibody; And

(iii) selecting the mutation of any variant with increased C1q-binding ability.

It relates to a method for identifying a mutation in an antibody that enhances the effector function of an antibody that binds to C1q, including.

In one embodiment, the at least one antibody is in the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W, such as E430G, E430S, E345K, and E345Q in the Fc region of a human IgG1 heavy chain. One or more amino acid substitution(s) selected from.

In another major aspect, the present invention

(i) preparing a variant of at least one parent antibody comprising a mutation in one or more amino acid(s) selected from the group corresponding to E430X, E345X, S440Y, or S440W in the Fc region of a human IgG1 heavy chain;

ii) evaluating the CDC-response induced by the variant when it binds to the surface of the antigen-expressing cell in the presence of effector cells or complement compared to the parent antibody; And

(iii) selecting mutations of any variant with increased CDC-response

It relates to a method of identifying a mutation in a parent antibody that increases the ability of the antibody to induce a CDC-reaction, comprising.

In one embodiment, the at least one antibody is E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of a human IgG1 heavy chain, such as E430G, E430S, E345K in the Fc region of a human IgG1 heavy chain. , And one or more amino acid substitution(s) selected from the group corresponding to E345Q.

Polypeptide of the invention

Parental polypeptide

As described herein, the present invention particularly relates to variants of the parental polypeptide comprising one or more mutations in the CH3 region of an immunoglobulin, for example an antibody heavy chain. The “parent polypeptide” may be a “parent antibody”. The "parent" antibody, which may be a wild-type antibody used as a starting material of the present invention before modification, can be produced, for example, by the hybridoma method first described in Kohler et al., Nature 256, 495 (1975). Or can be produced by recombinant DNA methods. Monoclonal antibodies are also described, for example, in Clackson et al., Nature 352, 624 628 (1991) and Marks et al., J. Mol. Biol. 222, 581 597 (1991)] can be used to isolate from phage antibody libraries. Monoclonal antibodies can be obtained from any suitable source. Thus, for example, monoclonal antibodies can be obtained from hybridomas prepared from murine spleen B cells obtained from mice immunized with, for example, cells expressing the antigen on the surface or with the antigen of interest in the form of a nucleic acid encoding the antigen of interest. I can. Monoclonal antibodies can also be obtained from hybridomas derived from antibody-expressing cells such as immunized human or non-human mammals such as rabbits, rats, dogs, primates, etc.

The parent antibody can be, for example, a chimeric or humanized antibody. In another embodiment, the antibody is a human antibody. Human monoclonal antibodies can be generated using transgenic or transchromosomal mice, such as HuMAb mice, that retain a portion of the human immune system rather than a mouse immune system. HuMAb mice have human immunoglobulin gene minilocus encoding unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, with targeted mutations that inactivate endogenous μ and κ chain loci. Contains (Lonberg, N. et al., Nature 368, 856 859 (1994)). Thus, mice show reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class conversion and somatic mutation to obtain high-affinity human IgG, κ monoclonal antibodies. (Lonberg, N. et al. (1994), supra; Lonberg, N. Handbook of Experimental Pharmacology 113, 49 101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol Vol. 13 65 93 (1995) and Harding, F. and Lonberg, N. Ann. NY Acad. Sci 764 536 546 (1995)). The preparation of HuMAb mice is described in Taylor, L. et al., Nucleic Acids Research 20, 6287 6295 (1992), Chen, J. et al., International Immunology 5, 647 656 (1993), Tuaillon et al., J. Immunol. 152, 2912 2920 (1994), Taylor, L. et al., International Immunology 6, 579 591 (1994), Fishwild, D. et al., Nature Biotechnology 14, 845 851 (1996). In addition, US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,789,650, US 5,877,397, US 5,661,016, US 5,814,318, US 5,874,299, US 5,770,429, US 5,545,807, WO 98/24884, WO 94/25585, WO 94/25585 See WO 92/22645, WO 92/03918 and WO 01/09187. Splenocytes from these transgenic mice can be used to generate hybridomas secreting human monoclonal antibodies according to well known techniques.

In addition, human antibodies of the invention or antibodies of the invention from other species may include, but are not limited to, phage display, retroviral display, ribosome display, mammalian display, yeast display, and other techniques known in the art. Can be identified through type techniques, and the resulting molecules can be subjected to further maturation, such as affinity maturation, such techniques are well known in the art. The specific strategy described in Example 17 can be applied to any antibody to make and obtain variants of the invention using phage-display.

Parental antibodies are not only limited to antibodies with natural, e.g., human Fc domains, but also mutations other than the mutations of the invention, such as e.g., affect glycosylation or make the antibody a bispecific antibody. It could be a capable mutation. The term “natural antibody” refers to any antibody that does not contain any genetically introduced mutations. Antibodies comprising naturally occurring modifications, eg different allotypes, should therefore be understood as “natural antibodies” in the sense of the present invention and may be understood as parent antibodies. Such antibodies can serve as a template for one or more mutations according to the invention, thus providing a variant antibody of the invention. Examples of parental antibodies containing mutations other than the mutations of the present invention promote the 1/2-molecular exchange of two antibodies comprising an IgG4-like CH3 region, and thus, bispecific, without involving the formation of aggregates. It is a bispecific antibody as described in WO2011/131746 (Genmab) using reducing conditions to form a red antibody. Other examples of parental antibodies include bispecific antibodies such as heterodimeric bispecific antibodies: triomab (pregenius); Bispecific IgG1 and IgG2 (Linat Neuroscience Corporation); FcΔAdp (regenerone); Knob-Into-Hall (Genentech); Electrostatic steering (Amgen, Chugai, Oncomed); Seed body (Merck); Azimetric scaffold (zymeworks); mAb-Fv (Zencor); And LUZ-Y (Genentech). Other exemplary parental antibody formats include, but are not limited to, wild type antibodies, full length antibodies or Fc-containing antibody fragments, human antibodies, or any combination thereof.

The parent antibody may bind to any target, and examples of such targets or antigens to which the present invention can act may be as follows, but are not limited thereto; 5T4; ADAM-10; ADAM-12; ADAM17; AFP; AXL; ANGPT2 anthrax antigen; BSG; CAIX; CAXII; CA 72-4; Carcinoma Associated Antigen CTAA16.88; CCL11; CCL2; CCR4; CCR5; CCR6; CD2; CD3E; CD4; CD5; CD6; CD15; CD18; CD19; CD20; CD22; CD24; CD25; CD29; CD30; CD32B; CD33; CD37; CD38; CD40; CD40LG; CD44; CD47; CD52; CD56; CD66E; CD72; CD74; CD79a; CD79b; CD80; CD86; CD98; CD137; CD147; CD138; CD168; CD200; CD248; CD254; CD257; CDH3; CEA; CEACAM5; CEACAM6; CEACAM8; Claudin4; CS-1; CSF2RA; CSPG-4; CTLA4; Cripto; DLL4; ED-B; EFNA2; EGFR; Endothelin B receptor; ENPP3; EPCAM; ERBB2; ERBB3; FAP alpha; Fc gamma RI; FCER2; FGFR3; Fibrin II beta chain; FLT1; FOLH1; FOLR1; FRP-1; GD3 ganglioside; GDF2; GLP1R; Glypican-3; GPNMB; HBV (hepatitis B virus); HCMV (human cytomegalovirus); Heat shock protein 90 homologue [Candida albicans]; Herpes simplex virus gD glycoprotein; HGF; HIV-1; HIV-1 IIIB gp120 V3 loop; HLA-DRB (HLA-DR beta); Human respiratory syncytial virus, glycoprotein F; ICAM1; IFNA1; IFNA1; IFNB1 bispecific; IgE Fc; IGF1R; IGHE connection area; IL12B; IL13; IL15; IL17A; IL1A; IL1B; IL2RA; IL4; IL5; IL5RA; IL6; IL6R; IL9; Interleukin-2 receptor beta subunit; ITGA2; ITGA2B ITGB3; ITGA4 ITGB7; ITGA5; ITGAL; ITGAV_ITGB3; ITGB2; KDR; L1CAM; Lewis-y; Lipid A, domain of lipopolysaccharide LPS; LTA; MET; MMP14; MMp15; MST1R; MSTN; MUC1; MUC4; MUC16; MUC5AC; NCA-90 granulocyte cell antigen; Nectin 4; NGF; NRP; NY-ESO-1; OX40L; PLAC-1; PLGF; PDGFRA; PD1; PDL1; PSCA; Phosphatidylserine; PTK-7; Pseudomonas aeruginosa serotype IATS O11; RSV (human respiratory syncytial virus, glycoprotein F); ROR1; RTN4; SELL; SELP; STEAP1; Shiga-Like Toxin II B subunit [Escherichia coli]; SLAM7; SLC44A4; SOST; Staphylococcus epidermidis lipoteichoic acid; T cell receptor alpha_beta; TF; TGFB1; TGFB2; TMEFF2; TNC; TNF; TNFRSF10A; TNFRSF10B; TNFRSF12A; TNFSF13; TNFSF14; TNFSF2; TNFSF7; TRAILR2; TROP2; TYRP1; VAP-1; And vimentin.

The parent antibody can be any human antibody of any isotype, for example IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, IgM, and IgD, optionally a human full length antibody, such as a human full length IgG1 antibody. The parent antibody may comprise a sequence according to any of SEQ ID NOs: 1, 2, 3, 4 and 5.

Monoclonal antibodies for use in the present invention, such as parent antibodies and/or variants, can be produced, for example, by the hybridoma method first described in Kohler et al., Nature 256, 495 (1975), or , Or can be produced by recombinant DNA methods. Monoclonal antibodies are also described, for example, in Clackson et al., Nature 352, 624-628 (1991) and Marks et al., J. Mol. Biol. 222, 581-597 (1991)] can be used to isolate from phage antibody libraries. Monoclonal antibodies can be obtained from any suitable source. Thus, for example, monoclonal antibodies can be obtained from hybridomas prepared from murine spleen B cells obtained from mice immunized with, for example, cells expressing the antigen on the surface or with the antigen of interest in the form of a nucleic acid encoding the antigen of interest. I can. Monoclonal antibodies can also be obtained from hybridomas derived from antibody-expressing cells such as immunized human or non-human mammals such as rats, dogs, primates, etc.

In one embodiment, the antibody is a human antibody. Human monoclonal antibodies directed against any antigen can be generated using transgenic or transchromosomal mice bearing a portion of the human immune system rather than a mouse immune system. Such transgenic and transchromosomic mice include mice referred to herein as HuMAb® mice and KM mice, respectively, and are collectively referred to herein as “transgenic mice”.

HuMAb® mice contain a human immunoglobulin gene minilocus encoding an unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences with targeted mutations that inactivate endogenous μ and κ chain loci (Lonberg , N. et al., Nature 368, 856-859 (1994)). Thus, mice display reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes produce high affinity human IgG, κ monoclonal antibodies through class conversion and somatic mutation. (Lonberg, N. et al. (1994), supra; Lonberg, N. Handbook of Experimental Pharmacology 113, 49-101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 13 65-93 (1995) and Harding, F. and Lonberg, N. Ann. NY Acad. Sci 764 536-546 (1995)). The preparation of HuMAb® mice is described in Taylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992), Chen, J. et al., International Immunology 5, 647-656 (1993), Tuaillon et al. ., J. Immunol. 152, 2912-2920 (1994), Taylor, L. et al., International Immunology 6, 579-591 (1994), Fishwild, D. et al., Nature Biotechnology 14, 845-851 (1996)]. Has been. In addition, US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,789,650, US 5,877,397, US 5,661,016, US 5,814,318, US 5,874,299, US 5,770,429, US 5,545,807, WO 98/24884, WO 94/25585, WO 94/25585 See WO 92/22645, WO 92/03918 and WO 01/09187.

HCo7, HCo12, HCo17 and HCo20 mice have JKD disruption of their endogenous light chain (kappa) gene (as described in Chen et al. , EMBO J. 12, 821-830 (1993)), of their endogenous heavy chain gene. CMD disruption (as described in Example 1 of WO 01/14424), and KCo5 human kappa light chain transgene (as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)). . In addition, Hco7 mice have HCo7 human heavy chain transgene (as described in US 5,770,429), HCo12 mice have HCo12 human heavy chain transgene (as described in Example 2 of WO 01/14424), HCo17 mice HCo17 human heavy chain transgene (as described in Example 2 of WO 01/09187), HCo20 mice have HCo20 human heavy chain transgene. The resulting mice express human immunoglobulin heavy chain and kappa light chain transgenes in a background that is homozygous for disruption of endogenous mouse heavy and kappa light chain loci.

In the KM mouse strain, the endogenous mouse kappa light chain gene is disrupted in a homozygous manner as described in Chen et al., EMBO J. 12, 811-820 (1993), and the endogenous mouse heavy chain gene is WO 01/09187 It was destroyed in a homozygous manner as described in Example 1. This mouse strain carries the human kappa light chain transgene KCo5 as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996). In addition, this mouse strain carries human heavy chain transchromosomes consisting of the chromosome 14 fragment hCF (SC20) as described in WO 02/43478. HCo12-Balb/C mice can be generated by crossing HCo12 to KCo5[J/K](Balb) as described in WO/2009/097006. Splenocytes from these transgenic mice can be used to generate hybridomas secreting human monoclonal antibodies according to well known techniques.

In addition, any antigen-binding region can be identified through display-type techniques including, but not limited to, phage display, retroviral display, ribosomal display, and other techniques using techniques well known in the art. Alternatively, it can be obtained from antibodies from other species, and the resulting molecule can be subjected to further maturation, such as affinity maturation, and such techniques are well known in the art (eg, Hoogenboom et al. ., J. Mol. Biol. 227, 381 (1991) (phage display), Vaughan et al., Nature Biotech 14, 309 (1996) (phage display), Hanes and Plucthau, PNAS USA 94, 4937-4942 (1997 ) (Ribosome display), Parmley and Smith, Gene 73, 305-318 (1988) (phage display), Scott TIBS 17, 241-245 (1992), Cwirla et al., PNAS USA 87, 6378-6382 (1990) , Russel et al., Nucl. Acids Research 21, 1081-1085 (1993), Hogenboom et al., Immunol. Reviews 130, 43-68 (1992), Chiswell and McCafferty TIBTECH 10, 80-84 (1992)], And US 5,733,743). When display technology is used to produce non-human antibodies, such antibodies can be humanized.

The mutation according to the present invention may be deletion, insertion or substitution of one or more amino acids, but is not limited thereto. The substitution of such amino acids may be with any naturally occurring or non-naturally occurring amino acid.

"Single-mutant"

All embodiments described herein with reference to a parent antibody, a first parent antibody or a second parent antibody also apply to other parental, first parental or second parental polypeptides comprising the Fc-domain and binding region of an immunoglobulin. It should be understood as possible.

The antibody or polypeptide variant according to the "single-mutant" aspect of the present invention is the EU index of a human IgG1 antibody with amino acids at the corresponding positions in the IgG2, IgG3, and IgG4 parental antibodies and "exemplary" and "preferred" amino acid substitutions. It includes mutations, typically amino acid substitutions, in one or more amino acid residue(s) shown in Table 1 listing each amino acid residue numbered according to. In IgG1, an IgG2 fragment corresponding to residues 126 to 326, an IgG3 fragment corresponding to residues 177 to 377, and an IgG4 fragment corresponding to residues 127 to 327 are shown in FIG. 2.

[Table 1]

As shown in Table 1, amino acid substitutions that resulted in an increase in cell lysis of Wien133 cells in Example 19 are included as "preferred substitutions".

In one aspect, the present invention relates to a variant of a parental polypeptide comprising an Fc domain and a binding region of an immunoglobulin, wherein the variant is E430G, E430S, E430F, E430T, E345K, E345Q, E345R in the Fc region of a human IgG1 heavy chain. , E345Y, and one or more mutation(s) selected from the group corresponding to S440W, provided that any addition that alters the binding of the variant to the neonatal Fc receptor (FcRn) as determined by the method disclosed in Example 34. Does not contain a mutation of the Fc domain.

In another aspect, the present invention relates to a variant of a parental polypeptide comprising an Fc domain and a binding region of an immunoglobulin, wherein the variant is E430G, E430S, E430F, E430T, E345K, E345Q, in the Fc region of a human IgG1 heavy chain. It contains one or more mutation(s) selected from the group corresponding to E345R, E345Y, and S440W, provided that the neonatal Fc receptor (FcRn) as measured by the change in absorbance at OD405 nm as determined by the method disclosed in Example 34 ) Does not contain any additional mutations in the Fc domain that increase or decrease the binding of the variant to more than 30%, such as more than 20%, 10%, or 5%.

In another aspect, the present invention relates to a variant of a parental polypeptide comprising an Fc domain and a binding region of an immunoglobulin, wherein the variant is E430G, E430S, E430F, E430T, E345K, E345Q, in the Fc region of a human IgG1 heavy chain. E345R, E345Y, and one or more mutation(s) selected from the group corresponding to S440W, provided that the apparent affinity of the parental antibody for the mouse neonatal Fc receptor (FcRn) as determined by the method disclosed in Example 34 The Fc does not contain any additional mutations that increase by more than a fold by more than 0.5 or that do not decrease the apparent affinity of the parent polypeptide or parent antibody for mouse FcRn by more than a fold 2.

In one embodiment, the one or more mutation(s) is selected from the group corresponding to E430G, E430S, E345K, and E345Q in the Fc region of a human IgG1 heavy chain.

In one embodiment, the variant does not contain any additional mutations in the Fc domain that alter the antibody dependent cell-mediated cytotoxicity (ADCC) of the variant.

In one embodiment, the variant does not contain any additional mutations in the Fc domain that alter the plasma clearance rate of the variant as determined in the method disclosed in Example 37.

In another embodiment, the variant increases or decreases the plasma clearance rate of the variant by a fold greater than 3.0, such as a fold 2.5, fold 2.0, fold 1.5, or fold 1.2, as determined by the method disclosed in Example 37. It does not contain any additional mutations in the Fc domain.

In one embodiment, the variant does not contain any additional mutations in the Fc domain that alter the serum half-life of the variant.

In one embodiment, the variant does not contain any additional mutations in the Fc domain that alter the target-independent fluid phase complement activation of the variant as determined by the method disclosed in Example 36.

In one embodiment, the variant does not contain any additional mutations in the Fc domain.

In one embodiment, the variant contains only 1 mutation.

In one embodiment, the variant polypeptide may be a variant antibody comprising the Fc domain and antigen-binding region of an immunoglobulin.

In one specific embodiment, the amino acid substitution is E345R.

As shown in the examples, CD38 antibodies HuMab-005 and -003 (as described in WO 2006/099875) and/or CD20 antibodies HuMab-7D8 and -11B8 (as described in WO 2004/035607) comprising one of these amino acid substitutions. As described) and variants of the rituximab and/or EGFR antibody HuMab-2F8 (as described in WO 2002/100348) showed higher C1q-binding, complement activation and/or CDC than wild-type HuMab 005 and 7D8, respectively. .

It is also to be understood that the variant may contain one of the mutations of the “exemplary substitutions” listed in Table 1. In addition, the variant may comprise more than one mutation, such as 2, 3, 4, 5 or 6 of any of the mutations listed in Table 1.

In addition to the indicated mutations, the variants may have any of the characteristics as described for the parent antibody. In particular, the variant may be a human antibody. Variants can be of any IgG subtype, in addition to mutations.

When binding to its antigen on the surface, cell membrane, virion or another particle of an antigen-expressing cell, or when the antigen associates with a virion, wherein, optionally, the antigen is contained in the protein coat or lipid envelope of the virion ), these antibody variants have increased compared to the parent antibody (i) antibody mediated CDC, (ii) antibody mediated complement activation, (iii) C1q-binding, (iv) oligomer formation, (v) Oligomer stability, or any combination of (i) to (v). In one embodiment of (iv) or (v), the oligomer is a hexamer. In one embodiment, the variant may also have an increased ADCC compared to the parental polypeptide or parental antibody. In a further embodiment, the variant maintains the same or similar plasma clearance rate compared to the parent polypeptide or parent antibody. In a further embodiment, the variant is increased or decreased by a fold greater than 3.0, such as fold 2.5, fold 2.0, fold 1.5, or fold 1.2, as determined in the method disclosed in Example 37 when compared to the parental polypeptide or parent antibody. It does not have a plasma clearance rate.

Without wishing to be bound by any particular theory, the effect arising from substituting an amino acid residue of the present invention for an amino acid at the indicated position results in, for example, the effect itself, or when directly contacting the Fc domain of another molecule. It may be related or mutated to directly interact with another Fc domain or indirectly affect the intermolecular Fc:Fc interaction. Thus, without being bound by theory, substitutions directly or indirectly enhance the binding strength between antibody molecules in oligomer form, thereby improving the stability of oligomer structures such as hexamer, pentameric, tetramer, trimer, or dimer structures. It is believed to enhance. For example, amino acid substitutions promote or enhance the formation of new intermolecular Fc:Fc bonds, such as, but not limited to, Van der Waals interactions, hydrogen bonding, charge-charge interactions, or aromatic stacking interactions. Or promoting increased entropy upon Fc:Fc interaction by release of water molecules. In addition, referring to Table 1, "exemplary substitutions" may be selected based on size and physicochemical properties that participate in or promote intermolecular Fc:Fc interactions or intramolecular interactions. "Preferred substitutions" can be selected based on the optimal size and physicochemical properties to participate in or stimulate intermolecular Fc:Fc interactions or intramolecular interactions.

In one embodiment, the variant may comprise an additional mutation selected from Table 1.

In one embodiment, the variant comprises a combination of two mutations in amino acid residues selected from the group corresponding to E345X/E430X, E345X/S440Y, E345X/S440W, E430X/S440Y, and E430X/S440W.

In any embodiment in which such mutations in at least two amino acids are included in the variant, the mutation may be present in each heavy chain of the variant, or one of the two may be included in one of the heavy chains, and the other in the other heavy chain. Can be included, or vice versa.

In one embodiment, the mutation at two amino acid residues is a deletion, insertion or substitution. Substitution of these amino acids may be by any naturally occurring or artificial amino acid.

The mutations according to the present invention may each be deletion, insertion or substitution of one or more amino acids, but are not limited thereto. The substitution of such amino acids may be with any naturally occurring or non-naturally occurring amino acid.

Thus, in one embodiment, the mutation in at least one amino acid residue is a deletion.

In another embodiment, the mutation at at least one amino acid residue is an insertion.

In another embodiment, the mutation in at least one amino acid residue is a substitution.

Exemplary specific combinations of mutations at two amino acid residues are E345R/E430T, E345R/S440Y, E345R/S440W, E345R/E430G, E345Q/E430T, E345Q/S440Y, E345Q/S440W, E430T/S440Y, and E430T/S440W. .

In addition to mutations in one or more amino acids according to embodiments of the invention, the IgG heavy chain may comprise additional mutations known in the art, for example mutations that further improve effector function. Such additional mutations include known mutations that enhance CDC, Fc-gamma receptor binding or FcRn-binding and/or improve Fc-gamma receptor-mediated effector function.

In one embodiment, the variant according to the invention comprises known CDC enhancing modifications, for example fragment exchange between IgG isotypes for the production of chimeric IgG molecules (Natsume et al., 2008 Cancer Res 68(10), 3863- 72); One or more amino acid substitutions in the hinge region (Dall'Acqua et al., 2006 J Immunol 177, 1129-1138) and/or in or near the C1q-binding site in the CH2 domain concentrated around residues D270, K322, P329, and P331. One or more amino acid substitutions (Idusogie et al., 2001 J Immunol 166, 2571-2575; Michaelsen et al., 2009 Scand J Immunol 70, 553-564 and WO 99/51642). For example, in one embodiment, the variant according to the invention further comprises any combination of amino acid substitutions S267E, H268F, S324T, S239D, G236A and I332E that provides enhanced effector function through CDC or ADCC. (Moore et al., 2010 mAbs 2(2), 181-189). Other Fc mutations that affect binding to the Fc-receptor (described in WO 2006/105062, WO 00/42072, US Pat. No. 6,737,056 and US Pat. No. 7,083,784) or physical properties of the antibody (described in WO 2007/005612 A1) It can also be used in the variant of the present invention.

In one embodiment, the variant according to the invention further comprises a modification that enhances Fc-gamma receptor binding and/or Fc-gamma receptor-mediated effector function. This modification was achieved by (i) a reduction in the amount of fucose in CH2 attached glycosylation (glyco-engineering) (Umana P, et al., Nat Biotechnol 1999; 17: 176-80; Niwa R, et al., Clin Cancer. Res 2004; 10: 6248-55.), and (ii) site-directed mutagenesis (protein-engineering) of amino acids in the hinge or CH2 region of the antibody (Lazar GA, et al., Proc Natl Acad Sci USA 2006 ; 103: 4005-10).

In one embodiment, the variant according to the invention is further engineered at the FcRn binding site, for example to extend the half-life (t1/2) of an IgG antibody. These modifications include (i) the N434A and T307A/E380A/N434A mutations (Petcova et al. Int Immunol. 2006 Dec;18(12):1759); (ii) Substitution with one or more alanine residues of Pro238, Thr256, Thr307, Gln311, Asp312, Glu380, Glu382, and Asn434 to improve FcRn binding (Shields RL, et al. J. Biol. Chem. 2001;276: 6591); And (iii) M252Y/S254T/T256E, M252W, M252Y, M252Y/T256Q, M252F/T256D, V308T/L309P/Q311S, G385D/Q386P/N389S, G385R/Q386T/ in IgG1, which increases the affinity for FcRn. Amino acid substitution selected from P387R/N389P, H433K/N434F/Y436H, N434F/Y436H, H433R/N434Y/Y436H, M252Y/S254T/T256E-H433K/N434F/Y436H or M252Y/S254T/T256E-G385R/Q386T/P385R And combinations of amino acid substitutions (Dall'Acqua et al., supra).

"Double-mutant"

All embodiments described herein with reference to a parent antibody, a first parent antibody or a second parent antibody also apply to other parental, first parental or second parental polypeptides comprising the Fc-domain and binding region of an immunoglobulin. It should be understood as possible.

As described above and further below, the present invention also relates to the "bi-mutant" aspect, wherein the two mutations individually reduce effector function, but together bring effector function to the level of the parent antibody. Recover. When used together, the specificity of the variants increases. Antibody variants according to the "double-mutant" aspect comprise two mutations, typically amino acid substitutions, in specific amino acid residue interaction pairs K439 and S440, K447 and 448, or K447, 448, and 449.

Thus, in one aspect, the present invention relates to a variant of a parental polypeptide comprising an Fc domain and a binding region of an immunoglobulin, wherein the variant is E430G, E430S, E430F, E430T, E345K, E345Q in the Fc region of a human IgG1 heavy chain. , A first mutation selected from the group corresponding to E345R, E345Y, S440Y, and S440W, such as E430G, E430S, E345K, or E345Q; And (i) amino acid residues corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain (however, when the mutation in S440 is not S440Y or S440W, and the first mutation is S440Y or S440W, the second mutation is a human IgG1 heavy chain. It is a mutation in the amino acid residue corresponding to K439 in the Fc region of),

(ii) amino acid residues corresponding to K447D/E or K447K/R/H and 448P in the Fc region of a human IgG1 heavy chain; or

(iii) a second mutation selected from the group corresponding to K447D/E in the Fc region of a human IgG1 heavy chain or corresponding to amino acid residues corresponding to K447K/R/H and 448K/R/H and 449P. Tables 2A and B show the "exemplary" and "preferred substitutions" for the "double-mutant" (Table A) and "mixed-mutant" (Table 2B) aspects.

[Table 2A]

[Table 2B]

In one embodiment, the variant is a first mutation selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, and E345Y in the Fc region of a human IgG1 heavy chain, and at amino acid residues corresponding to K439 and S440. A second mutation of, provided that the mutation in S440 is not S440Y and S440W.

Variants may also contain only one of the amino acid residue substitutions, such as K439E or S440K, e.g., it is contemplated by the present invention that the variant optionally includes a mutation at K439 without the presence of a mutation at S440.

In one embodiment, the invention relates to variants in which the mutation at K439 is an amino acid substitution with an amino acid selected from E and D, such as K439E.

In another embodiment, the variant optionally comprises a mutation in S440 without the mutation in K439.

In one embodiment, the invention relates to variants in which the mutation at S440 is an amino acid substitution with an amino acid selected from K and R, such as S440K.

In one embodiment, the variant comprises a mutation in both K439 and S440.

In another embodiment, the mutation at K439 is selected from K439→D, E or R, such as K439D/E, and the mutation at S440 is selected from S440→D, E, K and R, such as S440K/R.

In another embodiment, the mutation at K439 is selected from K439D and K439E, and the mutation at S440 is selected from S440K and S440R.

In another embodiment, the variant comprises the K439E and S440K mutations.

In one embodiment, the parental polypeptide is a parental antibody comprising an Fc domain and an antigen-binding region of an immunoglobulin.

As described in Examples 4-6, antibody variants comprising only one of the K439E and S440K mutations have a sharply increased K _D for C1q, which reflects reduced complement activation and/or CDC capacity. Surprisingly, it was found that antibody variants of HuMAb 7D8 or 005 containing both mutations have recovered or increased C1q-binding or CDC. Without being limited to any particular theory, the underlying mechanism can be explained by each mutation sterically complementing each other, perhaps as illustrated in FIGS. 4 and 5.

In one embodiment, the parent polypeptide, and variants thereof, can be an antibody comprising the Fc domain and antigen-binding region of an immunoglobulin.

In another embodiment, a variant comprising a mutation at both positions K439 and S440 as described herein is compared to a parent antibody or an antibody variant comprising a mutation in only one of K439 and S440, complement dependent cytotoxicity (CDC ), C1q-binding, complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC), Fc-receptor binding including Fc-gamma receptor binding, protein A-binding, protein G-binding, antibody-dependent cell phagocytosis (ADCP), complement-dependent cellular cytotoxicity (CDCC), complement-enhancing cytotoxicity, opsonization, Fc-containing polypeptide internalization, target downregulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and its It shows an increase in Fc-mediated effector function selected from any combination.

The invention also compares the parent antibody (which may be a wild-type antibody) or an antibody variant comprising only one of the K439E or S440K mutations, (i) CDC mediated by the antibody, (ii) by the antibody. K439E and S440K mutations in the antibody to restore one or more of mediated complement activation, (iii) C1q-avidity, (iv) oligomer formation, (v) oligomer stability, or any combination of (i) to (v). Provides the use of. In one embodiment of (iv) or (v), the oligomer is a hexamer.

In one embodiment, the variant is selected from monospecific antibodies, bispecific antibodies or multispecific antibodies.

Mixed mutant

All embodiments described herein with reference to a parent antibody, a first parent antibody or a second parent antibody also apply to other parental, first parental or second parental polypeptides comprising the Fc-domain and binding region of an immunoglobulin. It should be understood as possible.

As described above, the inventors have also found that there are mutations that reduce effector function alone but restore effector function when used together, for example mutations at positions K439 and S440 in the Fc region of a human IgG1 heavy chain. . This concept can also be used to ensure pairing of two different antibodies by introducing K439 to one antibody and S440 to the other. Thus, antibody variants according to the "mixed-mutant" aspect typically include mutations other than those resulting in reduced or significantly reduced Fc:Fc interactions between the same Fc-molecules. However, as "mixed-mutant" antibodies, the variants of the invention pair with each other, for example recovered or even increased for a specific antibody variant pair compared to each variant alone or to a parent antibody or mixture of parent antibodies. CDC, C1q-linking, complement activation, oligomer formation, and/or oligomer stability. In one embodiment of the invention, the oligomer is a hexamer. In one embodiment, the antibody variant pair is additionally or alternatively maintained or improved other effector functions, such as C1q-binding, complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC), FcRn-binding, Fc -Fc-receptor binding, including gamma receptor-binding, protein A-binding, protein G-binding, antibody-dependent cellular phagocytosis (ADCP), complement-dependent cellular cytotoxicity (CDCC), complement-enhancing cytotoxicity, op Sonification, Fc-containing polypeptide internalization, target downregulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof. This aspect of the invention provides many uses in which selectivity as well as the strength of C1q-binding, complement activation, CDC or other effector functions can be modulated.

Exemplary mutation sites for each antibody variant in the “mixed-mutant” pair are shown in Table 2B. Specifically, the present invention provides a variant of an antibody comprising an antigen-binding region and an Fc-domain of an immunoglobulin, wherein the variant is a mutation at a residue in the Fc region of a human IgG1 heavy chain corresponding to one of K439 and S440 Includes.

In one embodiment, the mutation is an amino acid substitution at K439 with an amino acid selected from E or D, such as K439E. In one embodiment, the mutation is an amino acid substitution with an amino acid selected from K or R at S440, such as S440K.

In one embodiment, the variant comprises only an amino acid mutation at the position corresponding to K439 and no amino acid mutation at the position corresponding to S440 in the Fc region of the IgG1 heavy chain.

In one embodiment, the variant comprises only the amino acid mutation at the position corresponding to S440, provided that the mutation at S440 is not S440Y or S440W, and comprises an amino acid mutation at the position corresponding to K439 in the Fc region of the IgG1 heavy chain. I never do that.

Thus, in one embodiment, the invention also provides a first mutation selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W in the Fc region of a human IgG1 heavy chain; And a second mutation in the amino acid residue corresponding to K439 in the Fc region of a human IgG1 heavy chain.

In another embodiment, the invention also provides a first mutation selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, and E345Y in the Fc region of a human IgG1 heavy chain; And a second mutation in the amino acid residue corresponding to S440 in the Fc region of a human IgG1 heavy chain, provided that the second mutation is not S440Y or S440W.

In one embodiment, the two above-described embodiments can be combined in terms of a “mixed-mutant” pair according to the invention.

Each variant in the “mixed-mutant” pair may further comprise a mutation of the amino acids listed in Table 1.

In one embodiment of the invention, the “mixed-mutant” pair comprises a first variant of the parent antibody and a second variant of the parent antibody,

Here, the first variant comprises a first Fc-domain and an antigen-binding region of an immunoglobulin, and the first variant is (i) E430X, E345X, S440Y, in the Fc region of a human IgG1 heavy chain, in addition to the mutation in K439. And a first mutation in one or more amino acid residue(s) selected from the group corresponding to S440W, such as E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W, and the Fc region of a human IgG1 heavy chain. A second mutation at the position corresponding to K439 in;

The second variant comprises a second Fc-domain and an antigen-binding region of an immunoglobulin, said second variant being (i) E430X and E345X, such as E430G, in the Fc region of a human IgG1 heavy chain, in addition to the mutation at S440, A first mutation in one or more amino acid residue(s) selected from the group corresponding to E430S, E430F, E430T, E345K, E345Q, E345R, and E345Y,

And (ii) a second mutation at a position corresponding to S440 in the Fc region of the IgG1 heavy chain, provided that the mutation at S440 is not S440Y or S440W.

Other exemplary “mixed-mutant” pairs may further include, but are not limited to, any of the following pairs: a first variant comprising mutation K447E and a second variant comprising mutation K447/P448; A first variant comprising the mutation K447E and a second variant comprising the mutation K447/K448/P449.

In one embodiment, the mutation is a deletion, insertion or substitution. The substitution of such amino acids may be with any naturally occurring or non-naturally occurring amino acid.

In one embodiment, the mutation is a deletion.

In another embodiment, the mutation is an insertion.

In another embodiment, the mutation is a substitution of an amino acid.

In certain embodiments, the first variant and/or the second variant comprises a mutation in one or more amino acid residue(s) selected from the group corresponding to E430G, E430S, E345K, and E345Q in the Fc region of a human IgG1 heavy chain.

For example, in one embodiment, one variant in the “mixed-mutant” pair comprises one of E430G, E430S, E345K or E345Q with a K439E mutation, and the other variant is one of E430G, E430S, E345K or E345Q. One with the S440K mutation, and thus increased and more specific C1q-binding, complement activation, CDC, oligomer formation, oligomer stability, and/or other effector-related functions such as ADCC, Fc-gamma receptor binding, protein A-binding, protein G-binding, ADCP, CDCC, complement-enhancing cytotoxicity, antibody mediated phagocytosis, internalization, apoptosis, binding of opsonized antibodies to complement receptors, and/or combinations thereof.

The “mixed-mutant” aspect also includes two variants comprising more than one of each of the mutations listed in Table 2A in the Fc region of the human IgG1 heavy chain, such as a first variant comprising the mutation S440K/K447E, and the mutation K439E/ A second variant comprising K447/P448; For example, it may include a first variant comprising the mutation K439E/K447E, and a second variant comprising the mutation S440K/K447/P448.

As described herein, variants within a “mixed-mutant” pair can be derived from the same or different parent antibodies. In addition, the "mixed-mutant" aspect can be used for bispecific or asymmetric antibodies. In addition, the first, second and third antibodies can bind different epitopes on the same or different targets.

In addition, the "mixed-mutant" aspect is a tumor expressing two specific tumor antigens by using a first antibody against a first antigen having a K439E mutation and a second antibody against a second antigen having a S440K or S440R mutation. CDC or other effector responses that act more specifically on cells can be provided. More specifically for tumor cells expressing at least two, such as 2, 3, 4, 5 or 6 specific tumor antigens, by using the "mixed-mutant" aspect, which optionally comprises 3 variants which are bispecific antibodies. A specifically directed CDC or other effector response can be provided.

In one embodiment of any “single-mutant”, “bi-mutant” and “mixed-mutant” aspect, the variant is selected from a monospecific antibody, a bispecific antibody or a multispecific antibody.

In any embodiment of the “mixed-mutant” aspect, the first, second and/or third variant may comprise the same or different mutations of any of the amino acid substitutions listed in Table 1.

Multispecific antibodies

All embodiments described herein with reference to a parent antibody, a first parent antibody or a second parent antibody also apply to other parental, first parental or second parental polypeptides comprising the Fc-domain and binding region of an immunoglobulin. It should be understood as possible.

It is to be understood that any of the embodiments of the "single-mutant", "dual-mutant" and "mixed-mutant" aspects described herein may be used in the multispecific antibody aspects described below.

Thus, in one embodiment, the variant is selected from monospecific antibodies, bispecific antibodies or multispecific antibodies.

In certain embodiments, the bispecific antibody has the format described in WO 2011/131746.

In one main aspect, the invention provides a first polypeptide comprising a first CH2-CH3 region and a first antigen-binding region of an immunoglobulin, and a second CH2-CH3 region and a second antigen-binding region of an immunoglobulin. It relates to a variant of the parent antibody that is a bispecific antibody comprising a second polypeptide comprising a second polypeptide, wherein the first and second antigen-binding regions bind to different epitopes on the same or different antigens, and the first and/or second 2 The CH2-CH3 region contains one or more mutation(s) selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W in the Fc region of a human IgG1 heavy chain,

The first polypeptide comprises an additional mutation at an amino acid residue selected from those corresponding to K409, T366, L368, K370, D399, F405, and Y407 in the Fc region of a human IgG1 heavy chain;

The second polypeptide comprises an additional mutation at an amino acid residue selected from those corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc region of a human IgG1 heavy chain, wherein the further mutation in the first polypeptide is Differs from further mutations in the second polypeptide.

In one embodiment, the mutation is a deletion, insertion or substitution. The substitution of such amino acids may be with any naturally occurring or non-naturally occurring amino acid.

The bispecific antibody of the present invention is not limited to a specific format, and may be any one described above and herein.

In one specific embodiment of the invention, (i) the first polypeptide comprises an additional mutation at the amino acid residue corresponding to K409 in the Fc region of a human IgG1 heavy chain (eg, K409R);

(ii) the second polypeptide comprises an additional mutation (eg, F405L) at the amino acid residue corresponding to F405 in the Fc region of a human IgG1 heavy chain; Or alternatively

(iii) the first polypeptide comprises an additional mutation (eg, F405L) at the amino acid residue corresponding to F405 in the Fc region of a human IgG1 heavy chain;

(iv) The second polypeptide comprises an additional mutation (eg, K409R) at the amino acid residue corresponding to K409 in the Fc region of a human IgG1 heavy chain.

In certain embodiments, the mutation in one or more amino acid residue(s) is selected from the group corresponding to E430G, E430S, E345K, and E345Q in the Fc region of a human IgG1 heavy chain.

Such bispecific antibodies according to the present invention can be generated as described in Example 22. In addition, the effect on CDC killing by the resulting heterodimeric protein can be tested by using an assay as described in Example 23.

Bispecific antibodies may comprise, for example, an antigen-binding region of a CD20 antibody and an antigen-binding region of a CD38 antibody, and amino acid substitutions of one or more amino acids listed in Tables 1 and/or 2A/B. Exemplary CD20-binding regions include those of ofatumumab (2F2), 7D8 and 11B8, and rituximab (WO 2005/103081) described in WO2004/035607, which is incorporated herein by reference in its entirety. Exemplary CD38-binding regions include those of 003 and daratumumab (005) described in WO2006/099875, which is incorporated herein by reference in its entirety.

In one embodiment, the bispecific antibody binds to different epitopes on the same or different targets.

In another embodiment, the first mutations in the first and second polypeptides can be the same or different.

In one embodiment of the "single-mutant", "dual-mutant", "mixed-mutant" and multispecific antibody aspects, the variant is human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM , Or an IgE antibody, optionally a human full length antibody, such as a human full length IgG1 antibody.

In terms of any "single-mutant", "dual-mutant", "mixed-mutant", and multispecific antibody, the C1q-binding of the antibody is determined according to the assay described in Example 4, and CDC Is determined according to the assay described in Examples 5, 6 or 10, and mutations are optionally determined by comparing C1q-binding in the ELISA assay according to Example 3 with the C1q-binding in the cell-based assay according to Example 4. Not present at amino acid residues directly involved in C1q-binding as determined, ADCC is determined according to the assay described in Example 12.

Additionally, the present invention provides formulations of any of the “single-mutants”, “dual-mutants”, “mixed-mutants” and multispecific antibody aspects or variants of the embodiments described above. The invention also provides compositions, such as pharmaceutical compositions, comprising variants of any of the “bi-mutant” aspects and embodiments described above. The present invention also provides the use of any of the above variants, agents, or compositions as medicaments.

The above "single-mutant", "dual-mutant", "mixed mutant" and multispecific antibody aspects of the present invention are in particular the underlined residues of the human IgG1 heavy chain constant region (Uniprot Accession No. P01857; SEQ ID NO: 1) It can be applied to human antibody molecules having an IgG1 heavy chain comprising P247 to K447, which are related fragments corresponding to 130 to 330.

The present invention can also be applied to antibody molecules having a human IgG2 heavy chain portion. Amino acid residues P247 to K447 of the IgG1 heavy chain correspond to the underlined residues 126 to 326 of the IgG2 heavy chain constant region (accession number P01859; SEQ ID NO: 2).

The present invention can also be applied to antibody molecules having a human IgG3 heavy chain portion. Amino acid residues P247 to K447 of the IgG1 heavy chain correspond to residues 177 to 377 of the IgG3 heavy chain constant region (Uniprot accession number P01860, SEQ ID NO: 3), underlined as follows.

The present invention can also be applied to antibody molecules having a human IgG4 heavy chain portion. Amino acid residues P247 to K447 of the IgG1 heavy chain correspond to the underlined residues 127 to 327 of the IgG4 heavy chain constant region (accession number P01859, SEQ ID NO: 4).

The invention can also be applied to antibodies having a human IgG1m(f) allogeneic heavy chain portion. IgG1m(f) allotype amino acid sequence (CH3 sequence is underlined)-SEQ ID NO: 5

The alignment of each segment of the IgG1, IgG2, IgG3, IgG4, and IgG1m(f) constant regions is shown in Figure 2. Thus, any mutations in the amino acids listed in Table 1 or Tables 2A and B will be introduced at their equivalent positions in IgG2, IgG3, IgG4, and/or IgG1m(f) defined by alignment to obtain a variant according to the invention. I can.

In one embodiment, the invention provides variants of full length IgG1, IgG2, IgG3, or IgG4 antibodies comprising one or more amino acid substitutions according to any aspect described above.

In terms of any "single-mutant", "dual-mutant", "mixed-mutant" and multispecific antibody, the Fc region of the IgG1 heavy chain is from residues 130 to 330 of SEQ ID NO: 1, residue 126 of SEQ ID NO: 2 To 326, residues 177 to 377 of SEQ ID NO: 3, or residues 127 to 327 of SEQ ID NO: 4.

In one embodiment, the parent antibody comprises a sequence selected from SEQ ID NO: 1-5, such as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

In one embodiment, the Fc region of the IgG1 heavy chain comprises the sequence of residues 130-330 of SEQ ID NO:1.

The parent antibody can be any parent antibody as described herein. In this context, the parent antibody is also intended to be a first parent and a second parent antibody.

In one embodiment, the parent antibody is a human IgG1, IgG2, IgG3 or IgG4, IgA1, IgA2, IgD, IgM or IgE antibody.

In one embodiment, the parent antibody is a human full length antibody, such as a human full length IgG1 antibody.

In one embodiment, the parent antibody, the first parent antibody and the second parent antibody are a human IgG1 antibody optionally comprising an Fc-region comprising SEQ ID NO: 1 or 5, e.g. IgG1m(za) or IgG1m(f) homologous It's a variant.

In one embodiment, the parent antibody is a human IgG2 antibody optionally comprising an Fc-region comprising SEQ ID NO: 2.

In one embodiment, the parent antibody is a human IgG3 antibody optionally comprising an Fc-region comprising SEQ ID NO: 3.

In one embodiment, the parent antibody is a human IgG4 antibody optionally comprising an Fc-region comprising SEQ ID NO: 4.

In certain embodiments of any "single-mutant", "dual-mutant", "mixed-mutant" and multispecific antibody aspect, the variant is SEQ ID NO: 1, except for mutations introduced according to the invention At least 70%, 72%, 74%, 76%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86% at amino acids P247 to K447 of 2, 3, 4, and 5 , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or amino acid sequences having a degree of identity of at least about 99% Include.

Thus, the variant may comprise a sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, excluding any mutations defined herein.

Any of the above "single-mutants", "dual-mutants", "mixed-mutants" and multispecific aspects according to the present invention may be understood to include the following embodiments.

In one embodiment, the first and/or second parent antibody is a monovalent antibody, heavy chain antibody, strand-exchange engineered domain (SEED), triomab, dual variable domain immunoglobulin (DVD-Ig), knob-into -Hole antibody, mini-antibody, dual-affinity retargeting molecule (Fc-DART or Ig-DART); LUZ-Y antibody, biclonic antibody, dual targeting (DT)-Ig antibody, two-in-one antibody, crosslinked Mab, mAb ² , CovX-body, IgG-like bispecific antibody, Ts2Ab, BsAb, It is an antibody fragment optionally selected from the group consisting of Hercules antibody, TvAb, ScFv/Fc fusion antibody, Scorpion, scFv fragment fused to Fc domain, and double scFv fragment fused to Fc domain.

In a further embodiment, both the first and second parent antibodies bind antigens expressed on the surface of human tumor cells.

In a further embodiment, the antigens for the first and second parental antibodies are erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD38, CD138, CXCR5, c-Met, HERV-enveloped protein, periostin, Bigh3, SPARC, BCR, CD79, CD37, EGFrvIII, L1-CAM, AXL, tissue factor (TF), CD74, EpCAM and MRP3 are individually selected from the group consisting of.

In a further embodiment, the first and second parent antibodies are fully human antibodies.

In a further embodiment, the antigens for the first and second parent antibodies are selected in any order from CD20 and CD38, and optionally the first and second parent antibodies are selected in any order from 7D8 and 005.

In a further embodiment, both the first antibody and the second antibody bind to an antigen expressed on the surface of a bacterial cell or virion.

In another embodiment, the bacterial cell is S. Aureus, S. Epidermides, S. Pneumoniae, Bacillus anthracis, Pseudomonas aeruginosa, Chlamydia trachomatis, E. Coli, Salmonella, Shigella, Yersinia, S. Typhimurium, Neisseria meningitides and Mycobacterium tuberculosis.

In a further embodiment, the first and second parent antibodies bind the same antigen.

In another embodiment, the first and second parent antibodies are the same antibody.

In another embodiment, the parent antibody is selected from 7D8 and 005.

Composition

All embodiments described herein with reference to a parent antibody, a first parent antibody or a second parent antibody also apply to other parental, first parental or second parental polypeptides comprising the Fc-domain and binding region of an immunoglobulin. It should be understood as possible.

The invention also relates to compositions comprising variants, wherein the parent antibody can be any variant and parent antibody as described herein. Specific aspects and embodiments will be described below. In addition, such variants can be obtained according to any method as described herein.

In one aspect, the invention relates to a composition comprising a first and a second variant of a parental polypeptide each comprising an Fc domain and a binding region of an immunoglobulin, wherein the first and/or second variant is a human IgG1 heavy chain And one or more mutation(s) selected from the group corresponding to E430X, E345X, S440Y and S440W in the Fc region of.

In one embodiment, the first and/or second variant is one or more mutations selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W in the Fc region of a human IgG1 heavy chain. Include(s).

In a preferred embodiment, the first and/or second variant comprises one or more mutations selected from the group corresponding to E430G, E430S, E345K, and E345Q in the Fc region of a human IgG1 heavy chain.

In one embodiment, the first and second variants comprise one or more mutation(s), which may both be the same or different.

In another embodiment, the first variant corresponds to E430X, E345X, S440Y, and S440W, such as E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W in the Fc region of a human IgG1 heavy chain. Contains one or more mutation(s) selected from the group,

The second variant is at amino acid residues selected from the group corresponding to E430X, E345X, S440Y, and S440W, such as E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W in the Fc region of a human IgG1 heavy chain. Does not contain one or more mutation(s) of.

In one embodiment, the composition comprises at least one molecule comprising at least a CH2-CH3 domain of an immunoglobulin and a variant according to the invention, wherein the molecule is E430X, E345X, S440Y, and in the Fc region of a human IgG1 heavy chain S440W, such as a mutation in one or more amino acid residue(s) selected from the group corresponding to E430G, E430S, E345K, and E345Q.

In embodiments, the described molecules may be referred to as “Fc-only molecules” and may further comprise, for example, a hinge region. However, this hinge region may not be included.

Compositions comprising an Fc-only molecule and any variant according to the invention can be applied for use in imaging diagnostic methods or to modulate the avidity of the variant upon binding to the cell surface.

The Fc-only molecule may further comprise an additional mutation at the amino acid residue corresponding to K439 and/or S440 in the Fc region of the human IgG1 heavy chain, provided that the mutation at S440 is not S440Y or S440W, and the first mutation When is S440Y or S440W, an additional mutation is at the amino acid residue corresponding to K439 in the Fc region of the human IgG1 heavy chain.

In another embodiment, (i) the first variant further comprises a mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain, and (ii) the second variant is at S440 in the Fc region of a human IgG1 heavy chain. It further comprises a mutation at the corresponding position, provided that the mutation is not S440Y or S440W; or

(i) and (ii) alternatively

(iii) the first variant further comprises a mutation at a position corresponding to S440 in the Fc region of a human IgG1 heavy chain, provided that the mutation is not S440Y or S440W;

(iv) The second variant may further comprise a mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain.

In one embodiment, the mutation at position K439 in the Fc region of a human IgG1 heavy chain is K439D/E and/or the mutation at position S440 in the Fc region of a human IgG1 heavy chain is S440K/R.

In a further embodiment, the invention relates to a composition as defined herein, wherein

(i) the first variant further comprises a prodrug,

(ii) the second variant comprises an activator for the prodrug on the first variant; or

(i) and (ii) alternatively

(iii) the second variant comprises a prodrug,

(iv) The first variant may contain an activator for the prodrug on the second variant.

The term “prodrug” is to be understood according to the present invention as a relatively non-cytotoxic drug precursor that must undergo chemical conversion, eg by metabolic processes, before becoming an active pharmacological (anticancer) agent. Examples of prodrugs and methods of making them are well known in the art. An example is an antibody combination comprising an enzyme-prodrug, wherein drug delivery is the binding of an antibody conjugated with a prodrug to its antigenic target(s) present on the same cell and conjugated with an activator for the prodrug. It is provided by binding of antibodies. This brings the prodrug and its activator close to each other, whereby the drug can be released locally, for example infiltrating surrounding cells and killing these cells. (Senter and Springer, 2001 Adv Drug Deliv Rev. 2001 Dec 31;53(3):247-64, Senter, 1994 FASEB J. 1990 Feb 1;4(2):188-93).

The term "activator of a prodrug" is to be understood as a molecule capable of converting a prodrug into an active drug according to the present invention. Examples of activators of prodrugs and methods of making them are well known in the art. An example of an activator may be an enzyme that behaves as a catalyst for the conversion of a prodrug into an active drug. (Senter and Springer, 2001 Adv Drug Deliv Rev. 2001 Dec 31;53(3):247-64, Senter, 1994 FASEB J. 1990 Feb 1;4(2):188-93).

In one embodiment, the first and/or second parental polypeptides are first and second parental antibodies each comprising an Fc domain and an antigen-binding region of an immunoglobulin.

In one embodiment, the first and second antibodies are each human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM, or IgE antibody, optionally each human full length antibody, such as each human full length IgG1 antibody.

In one embodiment, the first and second antibodies are each selected from monospecific, bispecific or multispecific antibodies.

In a further embodiment, the first and/or second parent antibody is a first polypeptide comprising a first CH2-CH3 region and a first antigen-binding region of an immunoglobulin, respectively, and a second CH2-CH3 region and a second A bispecific antibody comprising a second polypeptide comprising an antigen-binding region, wherein the first and second antigen-binding regions bind to the same antigen or different epitopes on different antigens, and the first CH2-CH3 region is Contains an additional amino acid mutation at a position selected from positions corresponding to K409, T366, L368, K370, D399, F405, and Y407 in the Fc region of a human IgG1 heavy chain; The second CH2-CH3 region comprises an additional amino acid mutation at a position selected from positions corresponding to F405, T366, L368, K370, D399, Y407, and K409 in the Fc region of a human IgG1 heavy chain, and the first CH2-CH3 region The further amino acid mutation at is different from the further amino acid mutation at the second CH2-CH3 region.

In a preferred embodiment, the additional amino acid mutation in the first CH2-CH3 region is at a position corresponding to K409 in the Fc region of a human IgG1 heavy chain (eg, K409R); A further amino acid mutation in the second CH2-CH3 region is at a position corresponding to F405 in the Fc region of a human IgG1 heavy chain (eg, F405L).

In one embodiment, the first and second variants of the composition bind different epitopes on the same or different antigens.

In one embodiment, one or both of the first variant and the second variant are conjugated to a drug, toxin or radiolabel, such as one or both of the first variant and the second variant are conjugated to the toxin via a linker.

In one embodiment, one or both of the first and second variants are part of the fusion protein.

In certain embodiments, the first and/or second variant of the composition comprises only one mutation.

In embodiments where the second variant does not comprise any of the listed mutations described herein, such second variant may include an example of any suitable second antibody listed above with respect to a method of increasing CDC. .

In one embodiment, at least one first mutation in the first and second variant is different.

In one embodiment, the first and second variants are respectively human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM or IgE antibodies, optionally each human full length antibody, such as each human full length IgG1 antibody.

In one embodiment, the first variant and the second variant are selected from monospecific antibodies, bispecific antibodies or multispecific antibodies.

In a further embodiment, the first and second variants bind different epitopes on the same antigen or on different antigens. Thus, in embodiments where the first and second antibodies are bispecific antibodies, the antibodies may each bind to two different epitopes. The at least two bispecific antibodies may be the same or different. When the bispecific antibodies are different, the composition thus targets up to four different epitopes on the same or different targets.

In another aspect, the invention relates to a composition comprising any of the variants, any bispecific antibodies or any compositions described herein, and a pharmaceutically acceptable carrier.

It is also contemplated that any embodiment according to the “mixed-mutant” aspect may be included in any composition embodiment.

In one embodiment, variants of the first and second parent antibodies bind antigens expressed on the same cell.

In another embodiment, the variant of the first parent antibody comprises an amino acid substitution of K439 with an amino acid selected from E and D.

In another embodiment, the amino acid substitution of the variant of the first parent antibody is K439E.

In another embodiment, the variant of the second parent antibody comprises an amino acid substitution of S440 with an amino acid selected from K and R.

In another embodiment, the amino acid substitution of the variant of the second parent antibody is S440K.

In another aspect, the invention relates to a pharmaceutical composition comprising a variant of a first parental polypeptide or parental antibody of any one of the embodiments listed above and a variant of a second parental polypeptide or parental antibody.

Pharmaceutical compositions can be formulated according to conventional techniques, such as those described in Remington: The Science and Practice of Pharmacy, 19 ^th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. Pharmaceutical compositions of the present invention can be used, for example, diluents, fillers, salts, buffers, detergents (e.g., nonionic detergents such as Tween-20 or Tween-80), stabilizers (e.g., sugar or protein -Free amino acids), preservatives, tonicity agents, antioxidants, tissue fixatives, solubilizers, and/or other substances suitable for inclusion in pharmaceutical compositions. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (e.g., glycerol, propylene glycol, polyethylene glycol).

The pharmaceutical composition can be administered by any suitable route and manner. In one embodiment, the pharmaceutical composition of the present invention is administered parenterally. As used herein, the term “parenterally administered” refers to a mode of administration other than enteral and topical administration, generally by injection, and epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, orbital Intracardiac, intradermal, intraperitoneal, intraperitoneal, transtraminal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intrathecal, intracranial, intrathoracic, epidural and intrasternal injections and infusions.

Kit of parts

All embodiments described herein with reference to a parent antibody, a first parent antibody or a second parent antibody also apply to other parental, first parental or second parental polypeptides comprising the Fc-domain and binding region of an immunoglobulin. It should be understood as possible.

The invention also relates to a kit of parts for simultaneous, separate or sequential use in therapy, including variants of the parent polypeptide and the parent antibody, wherein the parent polypeptide and any variant of the parent antibody are as described herein. I can. Specific aspects and embodiments will be described below. In addition, such variants can be obtained according to any of the methods described herein.

In one aspect, the invention relates to a kit of parts for simultaneous, separate or sequential use in therapy, comprising a first variant of the parent polypeptide and a second variant of the parent polypeptide, wherein the first variant is a human IgG1 heavy chain E430X, E345X, S440Y, and S440W, such as E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and one or more mutation(s) selected from the group corresponding to S440W in the Fc region of, Provided that the variant does not contain any additional mutations in the Fc domain that alter the binding of the variant to the neonatal Fc receptor (FcRn),

(i) the first variant comprises a mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain, the second variant comprises a mutation at a position corresponding to S440 in the Fc region of a human IgG1 heavy chain, , Provided that the mutation in S440 is not S440Y or S440W,

(ii) The first variant contains a mutation at a position corresponding to K447D/E in the Fc region of a human IgG1 heavy chain, and the second variant corresponds to K447K/R/H and 448P in the Fc region of a human IgG1 heavy chain. Contains a mutation at the position of, or

(iii) the first variant contains a mutation at a position corresponding to K447D/E in the Fc region of a human IgG1 heavy chain, and the second variant is K447K/R/H, 448K/R in the Fc region of a human IgG1 heavy chain Include mutations at positions corresponding to /H and 449P.

In one embodiment, one or both of the first variant of the parent polypeptide and the second variant of the parent polypeptide may be an antibody comprising the Fc domain and antigen-binding region of an immunoglobulin.

In one embodiment, the mutation at the position corresponding to K439 in the Fc region of a human IgG1 heavy chain is K439D/E and/or the mutation at the position corresponding to S440 in the Fc region of a human IgG1 heavy chain is S440K/R.

In another aspect, the present invention comprises a first variant of a parent polypeptide comprising the Fc-domain and binding region of an immunoglobulin and a second variant of the parent polypeptide comprising the Fc-domain and binding region of an immunoglobulin. , To a kit of parts for simultaneous, individual or sequential use in therapy, wherein

Variants include one or more mutation(s) selected from the group corresponding to E430X, E345X, S440Y, and S440W, such as E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W in the Fc region of a human IgG1 heavy chain. ), provided that the Fc domain does not contain any additional mutations that alter the binding of the variant to the neonatal Fc receptor (FcRn),

The second variant is at amino acid residues selected from the group corresponding to E430X, E345X, S440Y, and S440W, such as E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y, and S440W in the Fc region of a human IgG1 heavy chain. Does not contain mutations of.

In embodiments where the second variant does not comprise any of the listed mutations described herein, such second variant may include an example of any suitable second antibody listed above with respect to the method of effector function.

In one embodiment, the at least one first mutation in the first and second variant is different.

In one embodiment, the first and second variants are respectively human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM or IgE antibodies, optionally each human full length antibody, such as each human full length IgG1 antibody.

In one embodiment, the first variant and the second variant are each selected from monospecific antibodies, bispecific antibodies or multispecific antibodies.

In a further embodiment, the first and second variants bind to the same antigen or to different epitopes on different antigens. Thus, in embodiments where the first and second antibodies are bispecific antibodies, the antibodies may each bind to two different epitopes. The at least two bispecific antibodies may be the same or different. Where the bispecific antibodies are different, the kit of parts for simultaneous, separate or sequential use in therapy thus comprises targeting up to four different epitopes on the same or different targets.

In a further embodiment, one or both of the first and second variants are conjugated to a drug, toxin or radiolabel, such as one or both of the first and second variants are conjugated to the toxin via a linker.

In a further embodiment, one or both of the first variant and the second variant are part of a fusion protein.

It is also contemplated that any embodiment according to the “mixed-mutant” aspect may be included in a kit embodiment of any parts for simultaneous, separate or sequential use in therapy.

In one embodiment, variants of the first and second parent antibodies bind antigens expressed on the same cell.

In another embodiment, the variant of the first parent antibody comprises an amino acid substitution of K439 with an amino acid selected from E and D.

In another embodiment, the amino acid substitution of the variant of the first parent antibody is K439E.

In another embodiment, the variant of the second parent antibody comprises an amino acid substitution of S440 with an amino acid selected from K and R.

In another embodiment, the amino acid substitution of the variant of the second parent antibody is S440K.

In another aspect, the invention provides for simultaneous, separate or sequential use in therapy, comprising a variant of a first parental polypeptide or parental antibody and a variant of a second parental polypeptide or parental antibody of any one of the embodiments listed above. It relates to a kit of pharmaceutical parts for.

Kits of pharmaceutical parts for simultaneous, separate or sequential use in therapy can be administered by any suitable route and manner. In one embodiment, the kit of pharmaceutical parts for simultaneous, separate or sequential use in the therapy of the invention is administered parenterally. The term "parenterally administered" as used herein refers to administration by administration other than enteral and topical administration, generally by injection, epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, Intraorbital, intracardiac, intradermal, intraperitoneal, intratenonal, transtraminal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intrathecal, intracranial, intrathoracic, epidural, and intrasternal injections and injections do.

Combination

In addition, the present invention provides a formulation of a variant of any of the “single-mutant” aspects or embodiments described above, ie formulations comprising multiple copies of the variant. The invention also provides compositions, such as pharmaceutical compositions, comprising variants of any of the “single-mutant” aspects and embodiments described above. The present invention also provides the use of any such "single-mutant" variant, agent, or composition as a medicament.

The present invention also provides a combination of variants in which one variant comprises at least one mutation according to the present invention, and one variant comprises at least one other mutation according to the present invention, as well as formulations and pharmaceutical compositions of such variant combinations, And its use as a medicament. Preferably, the two variants bind the same or different antigens, typically expressed on the surface of the same cell, cell membrane, virion and/or different particle.

Conjugate

All embodiments described herein with respect to a parent antibody, a first parent antibody or a second parent antibody may also be applicable to other parental, first parental or second parental polypeptides comprising the Fc-domain and binding region of an immunoglobulin. It should be understood as possible.

In one aspect, the present invention relates to a variant, wherein the variant is conjugated to a drug, toxin or radiolabel, such as the variant is conjugated to the toxin via a linker.

In one embodiment, the variant is part of a fusion protein.

In another aspect, the variant of the invention is not conjugated at the C-terminus to another molecule, such as a toxin or label. In one embodiment, the variant is conjugated to another molecule at another site, typically at a site that does not interfere with oligomer formation. For example, antibody variants can be linked at different sites to a compound selected from the group consisting of toxins (including radioisotopes), prodrugs or drugs. Such compounds can make the killing of target cells more effective, for example in cancer therapy. The resulting variant is thus an immunoconjugate.

Thus, in a further aspect, the invention provides antibodies linked or conjugated to one or more therapeutic moieties, such as cytotoxins, chemotherapy drugs, cytokines, immunosuppressants, and/or radioisotopes. Such conjugates are referred to herein as “immunoconjugates” or “drug conjugates”. Immunoconjugates comprising one or more cytotoxins are referred to as “immunotoxins”.

Cytotoxins or cytotoxic agents include any agent that is detrimental to (eg, kills) cells. The therapeutic agent suitable for the formation of the immunoconjugate of the present invention is Taxol, Cytocalacin B, Gramicidine D, Ethidium bromide, Emethine, Mitomycin, Etoposide, Tenofoside, Vincristine, Vinblastine, Colchicine, Doxorubicin , Daunorubicin, dihydroxy anthraxin dione, maytansine or analogs or derivatives thereof, enediene anti-tumor antibiotics, such as neocarzinostatin, calacheamycin, esperamicin, dinemycin, idamycin, Kedarcidin or its analogs or derivatives, anthracycline, mitoxantrone, mitramicin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, and puromycin, anti-metabolic Water (e.g. methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agent (E.g., mechloretamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dakar Bazin (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives such as carboplatin; as well as duocarmycin A, duocarmycin SA, CC-1065 (also known as raquelmycin), or CC- 1065 analogs or derivatives), dolastatin, pyrrolo[2,1-c] [1,4] benzodiazepine (PDB) or analogs thereof, antibiotics (eg dactinomycin (formerly actinomycin)), bleomycin, Daunorubicin (formerly Daunomycin), Doxorubicin, Idarubicin, Mitramycin, Mitomycin, Mitoxantrone, Plicamycin, Anthramycin (AMC)), antimitotic agents (e.g., tubulin- Inhibitors) such as monomethyl auristatin E, monomethyl auristatin F, or other analogs or derivatives of dolastatin 10; Histone deacetylase inhibitors, such as hydroxamic acid tricostatin A, vorinostat (SAHA), velinostat, LAQ824, and panobinostat as well as benzamide, entinostat, CI994, mothinostat and aliphatic acid compounds, For example phenylbutyrate and valproic acid, proteasome inhibitors such as danoprevir, bortezomib, amatoxins such as α-amantine, diphtheria toxin and related molecules (eg, diphtheria A chain and active fragments thereof and hybrid molecules); Lysine toxin (e.g., lysine A or deglycosylated lysine A chain toxin), cholera toxin, shiga-like toxin (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, Tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, allorin, saporin, modecin, gellanine, abrin A chain, modecin A chain, alpha-sarcin, alleuri Aleurites fordii protein, diantin protein, Phytolacca americana protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, cursin, crotin, sapona Saponaria officinalis inhibitors, gelonin, mitogelin, restrictocin, phenomycin and enomycin toxins. Other suitable conjugation molecules include antimicrobial/soluble peptides such as CLIP, maginin 2, melittin, cecropin and P18; Ribonuclease (RNase), DNase I, Staphylococcus enterotoxin-A, U.S. antiviral protein, diphtherine toxin and Pseudomonas endotoxin. See, for example, Pastan et al. , Cell 47, 641 (1986) and Goldenberg, Calif. A Cancer Journal for Clinicians 44, 43 (1994). Therapies that can be administered in combination with an antibody of the invention as described elsewhere herein, such as, for example, anticancer cytokines or chemokines, are also candidates for therapeutic moieties useful for conjugation to the antibodies of the invention.

In one embodiment, a drug conjugate of the invention comprises an antibody as disclosed herein conjugated to an auristatin or auristatin peptide analogue and derivative (US5635483; US5780588). Auristatin interferes with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584), anticancer (US5663149) and antifungal It was found to have activity (Pettit et al., (1998) Antimicrob. Agents and Chemother. 42:2961-2965). The auristatin drug moiety can be attached to the antibody through a linker, through the N (amino) terminus or C (terminus) of the peptidic drug moiety.

Exemplary auristatin embodiments include the N-terminal-linked monomethyl auristatin drug moieties DE and DF (Senter et al., Proceedings of the American Association for Cancer Research. Volume 45, abstract number 623 ] (Presented on March 28, 2004) and described in US 2005/0238649)

An exemplary auristatin embodiment is MMAE (monomethyl auristatin E). Another exemplary auristatin embodiment is MMAF (monomethyl auristatin F).

In one embodiment, an antibody of the invention comprises a conjugated nucleic acid or nucleic acid-associated molecule. In one such embodiment, the conjugated nucleic acid is a cytotoxic ribonuclease, an antisense nucleic acid, an inhibitory RNA molecule (eg, siRNA molecule) or an immunostimulatory nucleic acid (eg, an immunostimulatory CpG motif-containing DNA molecule). In another embodiment, the antibodies of the invention are conjugated to an aptamer or ribozyme.

In one embodiment, an antibody is provided comprising one or more radiolabeled amino acids. Radiolabeled variants can be used for both diagnostic and therapeutic purposes (conjugation to a radiolabeled molecule is another possible feature). Non-limiting examples of labels for polypeptides include 3H, 14C, 15N, 35S, 90Y, 99Tc, and 125I, 131I, and 186Re. Methods for the preparation of radiolabeled amino acids and related peptide derivatives are known in the art (for example, Junghans et al., in Cancer Chemotherapy and Biotherapy 655-686 (2 ^nd Ed., Chafner and Longo, eds). ., Lippincott Raven (1996)) and US 4,681,581, US 4,735,210, US 5,101,827, US 5,102,990 (US RE35,500), US 5,648,471 and US 5,697,902). For example, radioactive isotopes can be conjugated by the chloramine-T method.

In one embodiment, variants of the invention are conjugated to a radioisotope or radioisotope-containing chelate. For example, the variant can be conjugated to a chelating agent linker, such as DOTA, DTPA or tiuxetane, which allows the antibody to form a complex with the radioisotope. In addition, variants may additionally or alternatively contain or be conjugated to one or more radiolabeled amino acids or other radiolabeled molecules. Radiolabeled variants can be used for both diagnostic and therapeutic purposes. In one embodiment, a variant of the invention is conjugated to an alpha-emitter. Non-limiting examples of radioisotopes include ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹²⁵ I, ¹¹¹ In, ¹³¹ I, ¹⁸⁶ Re, ²¹³ Bs, ²²⁵ Ac and ²²⁷ Th.

In one embodiment, the variant of the invention is IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, From the group consisting of IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNα, IFNβ, IFNγ, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ansestim, and TNFα It can be conjugated to a cytokine of choice.

Variants of the present invention may also be chemically modified, for example by covalent conjugation to a polymer to increase its circulating half-life. Exemplary polymers and methods of attaching them to peptides are illustrated, for example, in US 4,766,106, US 4,179,337, US 4,495,285 and US 4,609,546. Additional polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., PEG having a molecular weight of about 1,000 to about 40,000, such as about 2,000 to about 20,000).

In order to conjugate the variant of the present invention to the molecule(s) to be conjugated, such as those described above, see Hunter et al., Nature 144, 945 (1962), David et al., Biochemistry 13, 1014 (1974), Pain et al. al., J. Immunol. Meth. 40, 219 (1981) and Nygren, J. Histochem. and Cytochem. 30, 407 (1982)], any method known in the art may be used. Such variants can be generated by chemically conjugating other moieties to the N-terminal side or C-terminal side of the variant or fragment thereof (e.g., antibody H or L chain) (see, for example, Antibody Engineering Handbook, edited by Osamu Kanemitsu, published by Chijin Shokan (1994)). In addition, such conjugated variant derivatives can be produced by contacting internal moieties or sugars where appropriate.

These agents can be coupled directly or indirectly to the variants of the invention. One example of an indirect coupling of a second agent is through a spacer or linker moiety to a cysteine or lysine residue in a bispecific antibody. In one embodiment, the variant is conjugated to a prodrug molecule that can be activated with a therapeutic drug in vivo through a spacer or linker. In some embodiments, the linker is cleavable under intracellular conditions, and cleavage of such linker releases the drug unit from the antibody into the intracellular environment. In some embodiments, a linker can be cleaved by a cleavable action present in the intracellular environment (eg, within a lysosome or endosome or vesicle). For example, the spacer or linker can be cleaved by tumor-cell associating enzymes or other tumor-specific conditions from which the active drug is formed. Examples of such prodrug technologies and linkers are described in WO02083180, WO2004043493, WO2007018431, WO2007089149, WO2009017394 and WO201062171 (Syntarga BV, et al. ). Suitable antibody-prodrug technology and duocarmycin analogs can also be found in US Pat. No. 6,989,452 (Medarex), which is incorporated herein by reference. Linkers are additionally or alternatively peptidyl linkers that are cleaved by intracellular peptidase or protease enzymes, including but not limited to, for example, lysosome or endosome proteases. In some embodiments, the peptidyl linker is at least 2 amino acids long or at least 3 amino acids long. Cleavage agents may include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release the active drug into target cells (see, e.g. Dubowchik and Walker, 1999 , Pharm. Therapeutics 83:67-123). In a specific embodiment, the peptidyl linker cleavable by the intracellular protease is a Val-Cit (valine-citrulline) linker or a Phe-Lys (phenylalanine-lysine) linker (e.g., US6214345 (doxorubicin with a Val-Cit linker) Synthesis of and different examples of Phe-Lys linkers are described)). Examples of structures of Val-Cit and Phe-Lys linkers include, but are not limited to, MC-vc-PAB, MC-vc-GABA, MC-Phe-Lys-PAB or MC-Phe-Lys-GABA described below. , Where MC is an abbreviation for maleimido caproyl, vc is an abbreviation for Val-Cit, PAB is an abbreviation for p-aminobenzylcarbamate, and GABA is an abbreviation for γ-aminobutyric acid. The advantage of using the intracellular proteolytic release of the therapeutic agent is that the agent is typically attenuated when conjugated, and the serum stability of the conjugate is typically high.

In another embodiment, the linker unit is not cleavable and the drug is released by antibody degradation (see US 2005/0238649). Typically, such linkers are substantially insensitive to the extracellular environment. As used herein, in terms of a linker, "substantially insensitive to the extracellular environment" means no more than 20%, typically no more than about 15%, more typically no more than about 10% in a sample of the variant antibody drug conjugate compound. , And even more typically about 5% or less, about 3% or less, or about 1% or less linkers are meant to be cleaved when the variant antibody drug conjugate compound is present in the extracellular environment (eg, plasma). Whether the linker is substantially insensitive to the extracellular environment is determined, for example, after incubation of the variant antibody drug conjugate compound with plasma for a predetermined period (e.g., 2, 4, 8, 16 or 24 hours), It can be determined by quantifying the amount of free drug present in plasma. Exemplary embodiments comprising MMAE or MMAF and various linker components have the following structure, wherein Ab means an antibody and represents a drug-load (or average number of cytotoxic or cytotoxic drugs per antibody molecule). p is 1 to about 8, for example p can be 4-6, such as 3-5, or p can be 1, 2, 3, 4, 5, 6, 7 or 8).

Examples in which a cleavable linker is combined with auristatin include MC-vc-PAB-MMAF (also referred to as vcMMAF) and MC-vc-PAB-MMAF (also referred to as vcMMAE), wherein MC is maleimido Caproyl is an abbreviation, vc is an abbreviation for a linker based on Val-Cit (valine-citruline), and PAB is an abbreviation for p-aminobenzylcarbamate:

Other examples include auristatin in combination with a non-cleavable linker, such as mcMMAF (mc (MC is the same as mc in this context) is an abbreviation for maleimido caproyl):

In one embodiment, the drug linker moiety is vcMMAE. The vcMMAE drug linker moiety and conjugation method are disclosed in WO2004010957, US7659241, US7829531, US7851437 and US 11/833,028 (Seattle Genetics, Inc.) (incorporated herein by reference), and the vcMMAE drug linker. The moiety is bound to the antibody at cysteine using a method similar to that disclosed in the literature.

In one embodiment, the drug linker moiety is mcMMAF. The mcMMAF drug linker moiety and conjugation method are disclosed in US7498298, US 11/833,954, and WO2005081711 (Seattle Genetics, Inc.) (incorporated herein by reference), and the mcMMAF drug linker moiety is a method similar to that disclosed in the literature. Is bound to the variant at cysteine.

In one embodiment, a variant of the invention is attached to a chelating agent linker, eg, tiuxetane, which allows the bispecific antibody to be conjugated to a radioisotope.

In one embodiment, each arm (or Fab-arm) of the variant is coupled directly or indirectly to the same one or more therapeutic moieties.

In one embodiment, only one arm of the variant is coupled directly or indirectly to one or more therapeutic moieties.

In one embodiment, each arm of the variant is directly or indirectly coupled to a different therapeutic moiety. For example, in embodiments wherein the variant is a bispecific antibody and is prepared by controlled Fab-arm exchange of two different monospecific antibodies as described herein, e.g., the first and the second antibody, said dual Specific antibodies can be obtained using monospecific antibodies conjugated or associated with different therapeutic moieties.

Additional uses

All embodiments described herein with respect to a parent antibody, a first parent antibody or a second parent antibody may also be applicable to other parental, first parental or second parental polypeptides comprising the Fc-domain and binding region of an immunoglobulin. It should be understood as possible.

In a further aspect, the present invention relates to the CDC-mediated killing of target cells (e.g., tumor, bacterial or fungal cells) or target organisms (e.g., viruses) or bacterial or viral infected cells for use in medicine. It relates to a variant of the present invention for use as a medicament for the treatment of a required disease or disorder. Examples of such diseases and disorders include, but are not limited to, cancer and bacterial, viral or fungal infections.

In another aspect, the invention relates to kits of the variants, bispecific antibodies, compositions and parts described herein for the treatment of diseases such as cancer.

In another aspect, the present invention relates to a method of treating a human comprising administering a kit of variants, compositions or parts described herein.

In another aspect, the present invention relates to a method of treating cancer in a human comprising administering a variant, composition or kit of parts.

"Treatment" refers to the administration of an effective amount of a therapeutically active compound of the present invention for the purpose of sedation, amelioration, arrest or eradication (healing) of a symptom or disease state.

“Effective amount” or “therapeutically effective amount” refers to an amount effective during this period at the dosage required to achieve the desired therapeutic result. A therapeutically effective amount of an antibody may vary depending on factors such as the disease state of the individual, age, sex and weight, and the ability of the antibody to elicit a desired response in the individual. In addition, a therapeutically effective amount is an amount that has a therapeutically beneficial effect greater than any toxic or adverse effect of the antibody or antibody portion.

In another aspect, the invention relates to the use of a kit of variants, compositions or parts according to any of the embodiments described herein in a diagnostic method.

In another aspect, the invention relates to a method of diagnosis comprising administering to at least a portion of the body of a human or other mammal a variant, composition or kit of parts according to any of the embodiments described herein.

In another aspect, the invention relates to the use of a variant, composition or kit of parts according to any of the embodiments described herein in imaging at least a portion of the body of a human or other mammal.

In another aspect, the invention relates to a method of imaging at least a portion of the body of a human or other mammal comprising administering a kit of variants, compositions or parts according to any of the embodiments described herein.

Without being bound by theory, the effective amount of a therapeutically active compound can be reduced when any “single-mutant” aspect or embodiment according to the invention is introduced into said therapeutically active compound.

Suitable antigens for cancer antibodies may be the same as those described herein. Examples 15-18 illustrate specific applications to provide enhanced and/or more specific complement activation or CDC of tumor cells. For example, an anti-tumor antibody according to the "single-mutant" aspect (eg, including the E345R mutation) can provide an enhanced CDC or ADCC, ADCP response of tumor cells. In addition, in variants of this method, mutations according to the "single-mutant" aspect, such as for example E345R, E430, or S440S/W, or any other mutations as listed in Table 1, are added to each antibody. Thus, it is possible to provide an enhanced CDC and/or ADCC response specifically directed against tumor cells expressing at least two antigens.

Antibodies suitable for bacterial infection include, but are not limited to, S. Targeting aureus, such as chimeric monoclonal IgG1 pagibaximab (BSYX-A110; Biosynexus), targeting lipoteic acid (LTA) embedded in the cell wall of Staphylococcus, And Baker (Nat Biotechnol. 2006 Dec;24(12):1491-3) and Weisman et al. (Int Immunopharmacol. 2009 May;9(5):639-44)] (both of which are incorporated by reference in their entirety). Example 14 contains the E345R mutation. Specific embodiments using aureus antibody variants are described. However, other mutations in Table 1, including but not limited to E430G and S440W, can be applied in a similar manner to enhance the CDC-mediated ability of antibodies to bacterial antigens.

Suitable antigens for viral or fungal infections can be any of those described herein.

In one embodiment, the antigen to which the variant binds is not human EphA2. In another embodiment, the variant is not derived from human EphA2 mAb 12G3H11 (described in Dall'Acqua et al., supra), which is incorporated herein by reference in its entirety. In another embodiment, the antigen to which the variant binds is not IL-9. In another embodiment, the variant is not derived from the Fa-hG1 or Fa-hG4 antibody described in WO2007005612, or any variant thereof, which is incorporated herein by reference in its entirety. In one embodiment, the antigen to which the variant binds is not HIV-1 gp120. In another embodiment, the variant is not derived from a b12 human IgG1K antibody directed against gp120.

In certain embodiments, the variant is from a bispecific parent antibody. The bispecific antibody may be of any isotype, such as for example IgG1, IgG2, IgG3, or IgG4, and may be a full length antibody or an Fc-containing fragment thereof. Exemplary methods of making bispecific antibodies are described in WO 2008/119353 (Genmab).

Dosage

All embodiments described herein with respect to a parent antibody, a first parent antibody or a second parent antibody may also be applicable to other parental, first parental or second parental polypeptides comprising the Fc-domain and binding region of an immunoglobulin. It should be understood as possible. The effective dosage and dosage regimen for the antibody will depend on the disease or condition to be treated and can be determined by one of ordinary skill in the art. Exemplary non-limiting ranges for therapeutically effective amounts of antibodies of the invention are about 0.1 to 100 mg/kg, such as about 0.1 to 50 mg/kg, for example about 0.1 to 20 mg/kg, such as about 0.1 to 10 mg. /kg, for example about 0.5, such as about 0.3, about 1, about 3, about 5, or about 8 mg/kg.

Antibody variants of the present invention can also be administered in combination with one or more complement factors or related components to enhance the therapeutic efficacy of the variant and/or supplement complement consumption. Such complement factors and related components include, but are not limited to, C1q, C4, C2, C3, C5, C6, C7, C8, C9, MBL, and factor B. Combination administration can be simultaneous, separate or sequential. In certain embodiments, the invention provides kits comprising a pharmaceutical composition comprising a variant of the invention, and at least one complement factor or related ingredient, in the same or different pharmaceutical composition, along with instructions for use.

The antibody variants of the invention may also be administered in combination therapy, ie in combination with other therapeutic agents associated with the disease or condition being treated. Thus, in one embodiment, the antibody-containing medicament is for combination with one or more additional therapeutic agents, such as cytotoxic agents, chemotherapeutic agents, or anti-angiogenic agents. These combination administrations can be simultaneous, separate or sequential.

In a further embodiment, the invention comprises administering to a subject in need of treatment or prevention of a disease, such as cancer, a therapeutically effective amount of a variant or pharmaceutical composition of the invention in combination with radiotherapy and/or surgery, For example, it provides a method of treating or preventing cancer.

Manufacturing method

All embodiments described herein with respect to a parent antibody, a first parent antibody or a second parent antibody may also be applicable to other parental, first parental or second parental polypeptides comprising the Fc-domain and binding region of an immunoglobulin. It should be understood as possible.

The invention also provides isolated nucleic acids and vectors encoding the variants according to any of the above-described aspects, as well as vectors and expression systems encoding the variants. Suitable nucleic acid constructs, vectors and expression systems for antibodies and variants thereof are known in the art and described in the Examples. In embodiments where the variant comprises a heavy chain (or an Fc-containing fragment thereof) as well as a light chain, the nucleotide sequences encoding the heavy and light chain portions may be present on the same or different nucleic acids or vectors.

The present invention also

a) providing a nucleotide construct encoding at least the Fc region of the heavy chain of the antibody variant according to any one of the aspects described above,

b) expressing the nucleotide construct in a host cell, and

c) recovering the antibody variant from the cell culture of the host cell.

It provides a method of producing in a host cell an antibody variant according to any one of the above-described aspects, including, wherein the variant comprises at least an Fc region of a heavy chain.

In some embodiments, the antibody is a heavy chain antibody. However, in most embodiments, the antibody will also contain a light chain, so the host cell also expresses a light chain-encoding construct on the same or a different vector.

Host cells suitable for recombinant expression of antibodies are known in the art and include CHO, HEK-293, PER-C6, NS/0 and Sp2/0 cells. In one embodiment, the host cell is a cell capable of Asn-linked glycosylation of a protein, eg a eukaryotic cell, such as a mammalian cell, eg a human cell. In a further embodiment, the host cell is a non-human cell that has been genetically engineered to produce a glycoprotein having human-like or human glycosylation. Examples of such cells are genetically modified Pichia pastoris (Hamilton et al., Science 301 (2003) 1244-1246; Potgieter et al., J. Biotechnology 139 (2009) 318-325) and genes. -Modified Lemna minor (Cox et al., Nature Biotechnology 12 (2006) 1591-1597).

In one embodiment, the host cell is a host cell that is unable to efficiently remove the C-terminal lysine K447 residue from the antibody heavy chain. See, for example, Liu et al. (2008) J Pharm Sci 97: 2426] (incorporated herein by reference), Table 2 lists a number of such antibody production systems, such as Sp2/0, NS/0 or transgenic mammary gland (goat), Here only partial removal of the C-terminal lysine is obtained. In one embodiment, the host cell is a host cell with an altered glycosylation mechanism. Such cells are described in the art, and can be used as host cells to produce antibodies with altered glycosylation by expressing the variants of the present invention. See, eg, Shields, R.L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1], as well as EP1176195; WO03/035835; And WO99/54342. Additional methods for generating engineered glycoforms are known in the art and are described in Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473], US6602684, WO00/61739A1; WO01/292246A1; WO02/311140A1; WO 02/30954A1; Potelligent™ technology (Biowa, Inc., Princeton, NJ); GlycoMAb™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); US 20030115614; Okazaki et al., 2004, JMB, 336: 1239-49, including, but not limited to.

The invention also relates to an antibody obtained or obtainable by the method of the invention described above.

In a further aspect, the invention relates to a host cell capable of producing the antibody variants of the invention. In one embodiment, the host cell is transformed or transfected with a nucleotide construct of the invention.

The invention is further illustrated by the following examples, which should not be regarded as a further limitation.

Example

Example 1

Design and generation of 7D8 mutants

The human monoclonal antibody HuMab-7D8 (described in WO 2004/035607) was used as a model antibody. It belongs to the group of human anti-CD20 IgG1 antibodies, including ofatumumab (HuMax-CD20, 2F2). These antibodies target a unique membrane-proximal epitope on the CD20 molecule and display a strong CDC.

To test the functional relevance of complement activation and oligomeric Fc-Fc interactions in CDC, amino acids in the hydrophobic patch at the Fc:Fc interface were mutated to potentially disrupt the interaction and CDC potency on the Fc-Fc side of 7D8. In the first set of mutants (Table 3), mutations were introduced to change the charge at selected positions based on the 1HZH crystal structure, and were described as exposed in the hydrophobic patch in the CH2-CH3 domain (Burton Mol Immunol 1985 Mar ;22(3):161-206).

From the first set of mutations, it was found that I253D and H433A induce the strongest effect on the loss of CDC by 7D8 (eg, Example 5). The 1HZH crystal structure shows that I253 and H433 bind to two different pockets on opposite Fc positions of the pairwise antibodies. Based on these data, a second set of mutations were synthesized around the I253 and H433 positions within the crystal structure to further study the importance of residues in the interface on the Fc:Fc side to CDC. A second set of mutations around the I253 and H433 positions that potentially destabilize the Fc:Fc interface and consequently CDC are listed in Table 4.

To rule out the possibility that disruption of the direct binding site for C1q would be responsible for the observed effect on CDC, a double mutant was created based on two single mutants that showed loss of CDC to His ability to recover from loss was tested. This principle is schematically illustrated in FIG. 1D. Double mutants are listed in Table 5, and structural representations are presented in Figures 4 and 5.

Mutants were prepared using a Quikchange site-directed mutagenesis kit (Stratagene, USA). Briefly, the full length plasmid DNA template encoding the 7D8 heavy chain with the IgG1m(f) allotype was cloned using forward and reverse primers encoding the desired mutation. The resulting DNA mixture was digested using DpnI to remove the source plasmid DNA, and E. It was used to transform coli. Mutant plasmid DNA isolated from the resulting colonies was confirmed by DNA sequencing (Agowa, Germany). Plasmid DNA mixtures encoding both heavy and light chains of the antibody were prepared essentially as described by the manufacturer using 293 pectin (Invitrogen, USA) using Freestyle HEK293F cells (Invitrogen, USA). )) was transiently transfected.

[Table 3]

[Table 4]

[Table 5]

Example 2

CD20 binding on cells by 7D8 mutants

Binding of purified antibody samples to CD20-positive cells was analyzed by FACS analysis. The first set of mutations (Table 3) were tested in Daudi cells, and the second set of mutations (Table 4) were tested in Raji cells. 10 ⁵ cells were subjected to serial dilutions of antibody preparation in RPMI1640/0.1% BSA for 30 min at 4° C. (range 0.04 to 10 μg/mL in 3-fold dilution for the first set on Daudi, and for the second set on Raji. Incubated at 50 μL in a polystyrene 96-well round-bottom plate (Greiner bio-one 650101) with 3-fold dilution ranging from 0.003 to 10 μg/mL). After washing twice in RPMI1640/0.1% BSA, cells were incubated at 4° C. for 30 min in 100 μL with secondary antibody. As a secondary antibody, fluorescein isothiocyanate (FITC)-conjugated rabbit-anti-human IgG (F0056, Dako, Glostrup, Denmark; 1/100) in all experiments on Daudi cells and 7D8 antibody Was used in experiments on Raji cells using. For experiments on Raji cells using purified 7D8 antibody, R-phycoerythrin (R-PE)-conjugated goat F(ab') ₂ anti-human kappa light chain (2062-09, SouthernBiotech ); 1/500) was used as a secondary antibody. The cells were then washed twice in PBS/0.1% BSA/0.02% azide, resuspended in 100 μL PBS/0.1% BSA/0.02% azide, and on FACS Cantoll (BD Biosciences). Analyzed. Binding curves were analyzed using non-linear regression (S-shaped dose-response with variable slope) using GraphPad Prism V5.01 software (GraphPad Software, San Diego, CA, USA). I did.

Binding of 7D8 antibody to Daudi cells was not affected by the introduction of a point mutation in the CH2-CH3 domain and was the same for all tested mutants and wild type 7D8. In addition, binding of the 7D8 antibody to Raji cells was not significantly affected by the introduction of a point mutation in the CH2-CH3 domain compared to wild-type 7D8 except for E345R. Reduced binding of IgG1-7D8-E345R was detected on CD20-positive Raji cells at test concentrations in excess of 0.3 μg/mL. Also for H433D and H433R, reduced binding was detected at the highest antibody concentration tested (10 μg/mL). Reduced binding by IgG1-7D8-E345R, H433D and H433R can be explained by masking of the epitope of the secondary antibody, since direct labeling of E345R and H433R produces similar or even increased binding to Daudi cells. to be. The increased avidity can be explained by the increased binding on the Fc-Fc side by E345R and H433R compared to wild-type IgG1-7D8.

The combination of the K439E and S440K mutations did not affect the binding of the 7D8 antibody to Raji cells, and was identical to the single mutant and wild type 7D8.

Example 3

C1q binding ELISA by 7D8 mutant

C1q binding by the 7D8 mutant was tested in ELISA, where the purified antibody was coated on a plastic surface, which resulted in random antibody multimerization. Collected human serum was used as the source of C1q.

A 96-well Microlon ELISA plate (Grainer, Germany) was coated with serial dilutions of antibody in PBS (range 0.58-10.0 μg/mL at 1.5-fold dilution) at 4° C. overnight. Plates were washed and blocked with 200 μL/well 0.5x PBS supplemented with 0.025% Tween 20 and 0.1% gelatin. While washing between incubations, the plate was collected 3% human serum (Sanquin, product # M0008) and 100 μL/well rabbit anti-human C1q (Dako, product # A0136, 1/4,000) at 37° C. for 1 hour And incubated sequentially with 100 μL/well porcine anti-rabbit IgG-HRP (Dako, P0399, 1:10,000) at RT for 1 hour and at RT for 1 hour as detection antibody. Color was developed for about 30 minutes using 1 mg/mL 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS; Roche, Mannheim, Germany). The reaction was stopped by addition of 100 μL 2% oxalic acid. Absorbance was measured at 405 nm in a microplate reader (Biotek, Winuskey, Vermont). The log transformed data was calculated by fitting sigmoidal dose-response curves with variable slopes using GraphPad Prism software. The EC ₅₀ values of the mutants were normalized plate to plate to wild type IgG1-7D8 and multiplied by the mean of all wild type IgG1-7D8 data.

As shown in Figure 6 and Table 6, the point mutations tested had minimal effect on Clq binding as measured by ELISA. For the IgG1-7D8-I253D mutant, somewhat less efficient Clq binding was measured in ELISA (higher EC ₅₀ values). The coating efficacy was tested for all antibodies and found to be similar for all antibodies.

[Table 6]

Example 4

C1q binding on cells by 7D8 mutants

The antibody is coated on the plastic surface to create an artificial static system of antibody binding and Fc-tail presentation. Thus, complement binding was also tested in a cell-based assay, where Clq binding to antibody-opsonized CD20-positive B cells was determined by FACS analysis. In experiments with set 1 mutants, Daudi or Raji cells were suspended on ice in 90 μL RPMI 1640 medium containing 10% FBS (2 x 10 ⁶ cells/mL). A series of concentrations of 10 μL of C1q (Complement Technologies, Tyler, TX) were added (final concentration ranges vary from 0-60 μg/mL and 0-140 μg/mL depending on maximum binding) . Then, 10 μL of purified antibody (10 μg/mL final concentration, ie, saturated condition) was added, and the reaction mixture was immediately transferred to a 37° C. water bath and incubated for 1 hour. In experiments with set 2 mutants, test mAb was added in large quantities to Daudi cells, then variable concentrations of C1q were added to aliquots, and the mixture was incubated as described above. Cells were washed 3 times with PBS/1% BSA and incubated with rabbit FITC-labeled anti-C1q antibody (DakoCytomation, 10 μg/mL) at room temperature for 30 minutes. Cells were washed with PBS/1% BSA and resuspended in PBS or fixed in 2% formaldehyde in PBS. Flow cytometry was performed on a FACSCalibur flow cytometer (BD Biosciences), and the average fluorescence intensity was converted to an equivalent soluble fluorescence molecule (MESF) using calibrated beads (Spherotech). Made it. The dissociation constant (K _D value) for binding of C1q to CD20-positive cells opsonized with the indicated 7D8 antibody was determined using SigmaPlot® software (Systat Software Inc., Washington). Calculated using. Mean K _D values were calculated from repeated binding experiments (4 times on Daudi cells, 3 times on Raji cells) and compared to the K _D values for C1q binding on cells opsonized with wild type 7D8 (Table 7 and Table). 8).

Set 1 mutants were tested in both Daudi and Raji cells and obtained the same results. Contrary to the C1q ELISA results, most of the tested mutants showed decreased C1q avidity (increased K _D ) in both antibody-opsonized Daudi (Table 7A) and Raji cells (Table 8). Compared to wild-type 7D8, IgG1-7D8-Q311A and H435A showed little to no reduction, I253A, I253Y and N434A showed more pronounced reduction, and I253D and H433A showed C1q avidity on opsonized Daudi or Raji cells. Showed a very dramatic decrease in. IgG1-7D8-H435R showed somewhat higher avidity (lower K _D ) than wild type 7D8 for Clq binding on both cell types, but was not significant.

Set 2 mutants were tested on Daudi cells. Compared to wild type 7D8, IgG1-7D8-E345R, E382R and H433R showed increased avidity on opsonized Daudi cells, reflected by lower K _D values (Table 7B). All other set 2 mutants showed reduced avidity compared to wild-type 7D8, where G385D, Y436D, Q438D, K439E and S440K showed sharply increased K _D values (Table 7B), and H433D and Y436C were reliable. The K _D value showed a sharply reduced binding to an unmeasurable degree.

The double mutant IgG1-7D8-K439E/S440K showed restored C1q binding on antibody-opsonized Daudi cells, while both single mutants showed reduced C1q binding compared to wild type 7D8. The avidity of the K439E/S440K double mutant was slightly increased compared to the wild type 7D8 (Table 7C). The mixture of single mutant IgG1-7D8-K439E and IgG1-7D8-K440E was able to completely recover C1q binding, which is comparable to the C1q binding of wild type 7D8 (Table 7C).

The mismatch between C1q binding unchanged in ELISA (Example 3) and C1q binding affected in cell-based assays by IgG1-7D8 mutants was tested CH3 involved in Fc:Fc interactions between antibody molecules. Although the position does not directly affect C1q binding, it has been shown to be an important determinant that affects the dynamic positioning of the antibody Fc-tail when bound on cells, thereby also affecting the strength of C1q binding.

[Table 7A]

[Table 7B]

[Table 7C]

[Table 8]

Example 5

C1q efficacy by 7D8 mutants in CDC assay on CD20-positive Raji cells

C1q efficacy using cells opsonized with the IgG1-7D8 mutant was tested in a CDC assay to investigate the effect of the observed change in C1q avidity on CDC activity. Thus, CDC assays were performed using C1q-depleted normal human serum supplemented with a set of defined concentrations of C1q. 0.1 x 10 ⁶ Raji cells were harvested in round-bottom 96-well plates (Nunc, Rochester, NY) at 10 μg/mL of purified antibody and serial concentrations of human C1q (0.005, 0.025, 0.1, 0.3, 1.0, 5.0, 30.0 μg/mL) and in RPMI1640 medium supplemented with 0.1% BSA for 15 min at RT to a total volume of 100 μL. Then, 25 μL of C1q-depleted serum (Quidel, San Diego, CA) was added and incubated at 37° C. in a water bath for 30 minutes or in an incubator for 45 minutes. After incubation, the reaction was stopped by placing the sample on ice. Cell lysis was determined in FACS using propidium iodide (PI, Sigma Aldrich, Zwindrecht, Netherlands) viable cell exclusion assay. The% lysis was determined as follows:% lysis = (number of PI pos cells/total number of cells) x 100%.

The lysis by wild-type 7D8 in the presence of 30 μg/mL C1q minus the lysis when C1q was not added was set to 100%. CH ₅₀ values (C1q concentration causing 50% dissolution) were calculated by fitting a sigmoidal dose-response curve on log-transformed data using GraphPad Prism software. The CH ₅₀ values of the mutants were normalized to wild type 7D8 (Table 9).

The data in Table 9 show that IgG1-7D8-Q311A, E382R and H435A showed no decrease in C1q efficacy according to the C1q avidity measurement; I253A, I253Y, G385D, N434A and Y436C show a significant decrease in C1q-efficacy; I253D, H310K, K322A, H433A, H433D, Y436D, Q438D, K439E and S440K show that they almost completely lose the ability to induce CDC at all C1q concentrations tested.

IgG1-7D8-H435R and H433R used C1q somewhat more efficiently, which resulted in a more efficient CDC than wild type 7D8. IgG1-7D8-E345R showed a dramatic increase in C1q efficacy, which resulted in significantly higher CDC lysis compared to wild type 7D8 (Table 9).

Figure 7 is a combination of the K439E and S440K mutations that cause loss of CDC as both single mutants, when the two mutations are combined in one molecule (K439E/S440K double mutant) or when two single mutants are combined (K439E + S440K mixture) shows that CDC is restored in C1q potency assay.

[Table 9]

Example 6

CDC with 7D8 mutants in CDC assay for CD20-positive cells

0.1 x 10 ⁶ cells in a round-bottom 96-well plate (Nunc, Rochester, NY) with 80 μL of antibody at a series concentration (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg/mL). Pre-incubate at RT on a shaker for 15 minutes in total volume. Subsequently, 20 μL normal human serum was added as a source of C1q (20% final concentration) and incubated for 45 minutes in a 37° C. incubator. The reaction was stopped by adding 30 μL ice-cold RPMI medium supplemented with 0.1% BSA. Cell lysis was determined on FACS using propidium iodide.

For CDC assays on Daudi cells, EC ₅₀ values (antibody concentration resulting in 50% lysis) were calculated by fitting sigmoidal dose-response curves on log transformed data using GraphPad Prism software. The EC ₅₀ values of the mutants were normalized to wild type 7D8 (Table 10 and Table 11).

Table 10 shows that on Daudi cells, IgG1-7D8-I253A, Q311A, E382R, H433R and H435A showed no difference in CDC compared to wild type 7D8; A significantly worse CDC (higher EC ₅₀ ) than wild type 7D8 was found for IgG1-7D8-I253D, I253Y, H310K, G385D, H433A, H433D, N434A, Y436C, Y436D, Q438D, K439E, S440K and I253D/H433A. , This induced CDC only at higher antibody concentrations; C1q binding deficient mutant IgG1-7D8-K322A included as a control almost completely lost the ability to induce CDC and did not reach EC ₅₀ at the tested concentrations; IgG1-7D8-H435R showed more efficient CDC than wild type 7D8 on Daudi cells. Importantly, according to the C1q potency CDC assay, E345R showed a significantly better CDC than wild type 7D8, where it had a 10-fold lower EC ₅₀ value on Daudi cells (Table 10). Figure 8 shows when the K439E and S440K mutations (both causing loss of CDC as single mutants) are combined in one molecule (K439E/S440K double mutant) or when two single mutants are combined ( K439E + S440K mixture) shows that CDC is recovered.

Table 11 shows that similar data were found for the IgG1-7D8 mutant on Raji cells.

[Table 10]

[Table 11]

Example 7

Ranking of 7D8 mutants according to their ability to induce CDC

For the 7D8 mutants tested, between C1q binding on Daudi cells (described in Example 4) and C1q efficacy assay on Raji cells (described in Example 5), and on Daudi cells with C1q binding and Daudi and Raji cells. A correlation was found between the CDC assay for (described in Example 6) (correlation data table 13). Thus, all tested 7D8 mutants were ranked according to their ability to induce CDC as shown in Table 12 using the K _D values of the C1q binding assay on Daudi cells.

[Table 12]

[Table 13]

Example 8

Design and generation of CD38 antibody 005 mutants

The human monoclonal antibody HuMab 005 is a fully human IgG1,κ antibody described in WO/2006/099875. Here, it was used as a model antibody for verification of the identified Fc mutations that enhance CDC activity. The tested mutations are listed in Table 14.

DNA constructs for different mutants were prepared and transiently transfected as described in Example 1, using the heavy chain of HuMab 005 with IgG1m(f) allotype as a template for the mutagenesis reaction.

[Table 14]

Example 9

CD38 binding on cells by HuMab-005 mutants

Binding of crude antibody samples to CD38-positive Daudi and Raji cells was analyzed by FACS analysis. 10 ⁵ cells in 100 μL in RPMI1640/0.1% BSA with serial dilutions of antibody preparation (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg/mL) in a polystyrene 96-well round-bottom plate. Incubated at 4° C. for 30 minutes. After washing twice in RPMI1640/0.1% BSA, cells were washed with 50 μL of FITC-conjugated rabbit F(ab') ₂ anti-human IgG (Cat. No. F0056; Dako; 1:150) at 4° C. for 30 min. During incubation. The cells were then washed twice in PBS/0.1% BSA/0.02% azide, resuspended in 100 μL PBS/0.1% BSA/0.02% azide, and analyzed on a FACS cantol (BD Biosciences). Binding curves were analyzed using GraphPad Prism V5.01 software. As a negative control, the supernatant of mock-transfected cells was used.

The binding of HuMab 005 to Daudi cells was not significantly affected by the introduction of point mutations in the CH2-CH3 domain. All tested antibodies bound to Daudi cells in a dose-dependent manner. Binding was similar to wild-type HuMab-005 in all tested mutants, except for 005-E345R, which showed somewhat reduced binding. However, without being bound by any theory, the lower binding will be the result of reduced binding by the secondary antibody, similar to IgG1-7D8-E345 in Example 2. The actual avidity by 005-E345R may be similar or even increased compared to 005-WT, but we were unable to confirm this due to the lack of directly labeled antibodies.

The binding of HuMab-005 to Raji cells was also not significantly affected by the introduction of point mutations in the CH2-CH3 domain. All tested antibodies bound to Raji cells in a dose-dependent manner. Maximum binding was similar to wild type 005 for the 005-I253D and H433A mutants, and lower for the combination of 005-E435R, K439E, S440K mutants and 005-K439E + 005-S440K. However, without being bound by any theory, the lower binding may be the result of reduced binding by the secondary antibody, similar to IgG1-7D8-E345R in Example 2 (masking of epitopes).

Example 10

CDC assay for CD38-positive cells with mutants of CD38 antibody 005

0.1 x 10 ⁶ Daudi or Raji cells in a round-bottom 96-well plate in a series of concentrations (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg/mL) of crude antibody and 100 μL of total Pre-incubate at RT on a shaker for 15 minutes by volume. Then, 25 μL normal human serum was added as a source of C1q (20% final concentration) and incubated for 45 minutes in a 37° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.

The CDC enhancing ability of the E435R mutation, which was found to enhance the CDC activity of both 7D8 and 005 antibodies on Daudi and Raji cells, was further analyzed using different concentrations of normal human serum (NHS) against Wien133 cells. 0.1 x 10 ⁶ Wien133 cells in a round-bottom 96-well plate with a series of concentrations (0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg/mL) of crude antibody. Pre-incubate at RT on a shaker for 15 minutes with a total volume of 50 μL. The NHS was then added as a source of C1q in a total volume of 100 μL to reach a final concentration of either 20% or 50% NHS. The reaction mixture was incubated for 45 minutes in a 37° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.

Identified mutations in the CH2-CH3 region that either lose or increase CDC activity against CD20 antibody 7D8 were found to have the same effect against 005 antibodies that recognize CD38. FIG. 9 shows that 005-I253D, H443A, K439E and S440K showed complete loss of CDC activity in both Daudi (FIG. 9A) and Raji (FIG. 9B) cells, whereas the 005-E345R mutant strongly enhanced CDC in both cell lines. It shows that it showed activity. Compared to the 7D8 data, the combination of 005-K439E + 005-S440K, both of which lost CDC as single mutants, restored CDC. Surprisingly, 005-E435R induced CDC much more strongly on Wien133 cells, where wild-type 005 was unable to induce death by CDC (FIG. 9C ). CDC killing by 005-E345R on Wien133 cells was observed at both 20% and 50% serum concentrations (FIG. 9C ). Also on Raji cells, 7D8-E345R and 005-E345R both showed enhanced CDC in vitro in 50% serum, where they showed similar efficacy as in 20% serum (FIG. 9D ).

Since the E345R mutation in the CH2-CH3 region enhanced CDC activity in both the tested CD20 antibody 7D8 and CD38 antibody 005, it is believed that the E345R mutation is a generic antibody modification that can be applied to induce or enhance CDC.

Example 11

IgG1 antibody containing CDC-enhancing mutation E345R is less sensitive to inhibition of CDC by Fc binding peptide DCAWHLGELVWCT than wild-type antibody.

It has been found that by mutating amino acid positions in the hydrophobic patch at the Fc:Fc interface of IgG, CDC efficacy is hindered or enhanced. The relevance of the interaction at the Fc-Fc interface in CDC efficacy, and thus possibly the formation of an oligomer (eg, hexameric ring) structure as observed with a b12 crystal structure, was further investigated. Thus, a 13-residue peptide (DCAWHLGELVWCT (SEQ ID NO: 7)) targeting the consensus binding site in the hydrophobic patch region on the surface of wild-type IgG Fc was used (Delano et al., Science 2000 Feb 18;287(5456):1279). -83). Indeed, as a conformation region prepared to interact with various distinct molecules (Delano et al., Science 2000 Feb 18;287(5456):1279-83), the identification of the consensus binding site on the surface of IgG Fc was determined by IgG1 b12. It is consistent with the identification of the core amino acids in the hydrophobic patch involved in the Fc-Fc interaction within the structure (Saphire et al., Science 2001 Aug 10;293(5532):1155-9). The interactions present in all binding interfaces are by a shared set of 6 amino acids (Met-252, Ile-253, Ser-254, Asn-434, His-435, and Tyr-436), and a shared backbone contact. Mediated (Delano et al., Science 2000 Feb 18;287(5456):1279-83). Thus, Fc binding peptides are expected to affect Fc-Fc interactions and consequently CDC efficacy.

0.1 x 10 ⁶ Daudi cells were pre-incubated on a shaker at room temperature for 10 minutes in round-bottom 96-well plates with 1.0 μg/mL of crude antibody in 75 μL. The Fc binding peptide DCAWHLGELVWCT at a series concentration of 25 μL (range 0.06-60 μg/mL final concentration) was added to the opsonized cells and incubated for 10 minutes on a shaker at RT. Then 25 μL NHS was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for 45 minutes. The reaction was stopped by adding 25 μL ice-cold RPMI medium supplemented with 0.1% BSA. 15 μL propidium iodide was added and cell lysis was determined by FACS analysis.

It was found that CDC mediated by wild type 005 (FIG. 10A) or 7D8 (FIG. 10B) was inhibited by the Fc-binding peptide DCAWHLGELVWCT in a dose-dependent manner. These competitive data again suggests an association of Fc-Fc interactions in hydrophobic patches of IgG in CDC efficacy. CDC-enhancing IgG1-005-E345R and IgG1-7D8-E345R mutants were both less susceptible to competition by Fc-binding peptides compared to their corresponding wild-type antibodies, indicating that the E345R mutations had stability of Fc-Fc interactions. And, consequently, CDC.

Example 12

Antibody-dependent cell-mediated cytotoxicity (ADCC) of CD38 expressing cells by variants of the CD38 antibody HuMAb 005

Daudi cells were harvested (5x10 ⁶ cells/ml), washed (twice in PBS, 1200 rpm, 5 min), 10% cosmic calf serum (CCS) (HyClone, Logan, Utah, USA) ) Was collected in 1 mL RPMI 1640 medium supplemented with 200 μCi ⁵¹ Cr (chromium-51; Amersham Biosciences Europe GmbH, Rosendal, The Netherlands). The mixture was incubated at 37° C. for 1 hour in a shaking water bath. After washing of the cells (twice in PBS, 1200 rpm, 5 minutes), the cells were resuspended in RPMI 1640 medium supplemented with 10% CCS, counted by trypan blue exclusion, and a concentration of 1×10 ⁵ cells/mL Diluted with.

On the other hand, peripheral blood mononuclear cells (PBMC) were prepared using standard Ficoll density centrifugation according to the instructions of the manufacturer (lymphocyte separation medium; Lonza, Verbier, France) to soften fresh leukocytes (Sanquin, Amsterdam, Netherlands). Isolated from. After the cells were resuspended in RPMI 1640 medium supplemented with 10% CCS, the cells were counted by trypan blue exclusion and concentrated to 1×10 ⁷ cells/mL.

For ADCC experiments, 50 μL ⁵¹ Cr-labeled Daudi cells (5,000 cells) were added to a total of 100 μL in RPMI medium supplemented with 10% CCS with 15 μg/mL CD38 antibody IgG1-005 or mutant IgG1-005-E345R. Pre-incubated in 96-well microtiter plates by volume. After 10 minutes at RT, 50 μL PBMC (500,000 cells) was added to make the effector to target ratio 100:1. Maximum cell lysis was determined by incubating 50 μL ⁵¹ Cr-labeled Daudi cells (5,000 cells) with 100 μL 5% Triton-X100. The amount of spontaneous lysis was determined by incubating 5,000 ⁵¹ Cr-labeled Daudi cells in 150 μL medium without any antibody or effector cells. The level of antibody-independent cell lysis was determined by incubating 5,000 Daudi cells without antibody with 500,000 PBMCs. Subsequently, the cells were incubated for 4 hours at 37° C. and 5% CO ₂ . To determine the amount of cell lysis, the cells were centrifuged (1200 rpm, 3 minutes), and 75 μL of the supernatant was transferred to a micron tube, and then released ⁵¹ Cr was counted using a gamma counter. The measured counts per minute (cpm) were used to calculate the percentage of antibody-mediated lysis as follows:

(cpm sample-cpm Ab-independent dissolution)/(cpm maximum dissolution-cpm spontaneous dissolution) x 100%

Table 15 shows the calculated EC ₅₀ values of IgG1-005-wt and IgG1-005-E345R in the ADCC assay performed. Four samples were tested. IgG1-005-E345R shows significantly lower EC ₅₀ values than IgG1-005-wt of all four tested samples.

[Table 15]

11 shows that compared to the wild-type antibody HuMab-005, the mutant IgG1-005-E345R was able to induce ADCC at lower concentrations, thus showing an enhanced efficacy of ADCC ability.

Example 13

FcRn binding and pharmacokinetic analysis of 7D8 mutants compared to wild type 7D8

The neonatal Fc receptor (FcRn) is responsible for the long plasma half-life of IgG by protecting it from degradation. After internalization of the antibody, FcRn binds to the antibody Fc region in the endosome, where the interaction is stable in a mild acidic environment (pH 6.0). When the environment is recycled to the neutral (pH 7.4) plasma membrane, the interaction is lost and the antibody is released back into the circulation. This affects the plasma half-life of IgG.

The ability of the 7D8 mutant IgG1-7D8-E354R to interact with FcRn from mice, cynomolgus monkeys and humans was tested in ELISA. All incubations were performed at room temperature. A 96 well plate was coated with 5 μg/mL (100 μL/well) of the recombinantly produced biotinylated extracellular domain of FcRn (mouse, human or cynomolgus) (FcRnECDHis-B2M-BIO), PBST + 0.2% BSA It was diluted within 1 hour. Plates were washed 3 times with PBST, wild type IgG1-7D8 or IgG1-7D8-E354R serially diluted 3 times (in PBST/0.2% BSA, pH 6.0) was added, and the plates were incubated for 1 hour. Plates were washed with PBST/0.2% BSA, pH 6.0. Goat-anti-human IgG(Fab'2)-HRP (Jackson Immuno Research, catalog number: 109-035-097) diluted in PBST/0.2% BSA, pH 6.0 was added, and the plate was Incubated for 1 hour. After washing, ABTS was added as a substrate and the plate was incubated for 30 minutes in the dark. Absorbance was read at 405 using an EL808 ELISA reader.

In this study, mice were housed in a barrier unit of a central laboratory animal facility (Utrecht, Netherlands) and maintained in a top filter mounted cage with unlimited provision of water and feed. All experiments were approved by the Utrecht University Animal Testing Ethics Committee.

To analyze the pharmacokinetics of 7D8 mutants in vivo, SCID mice (CB-17/IcrCrl-scid-BR, Charles-River) 100 μg (5 mg/kg) wild type 7D8, IgG1-7D8- E354R, -S440K or K322A (3 mice per group) was injected intravenously.

50 μL blood samples were collected from the saphenous vein 10 minutes, 4 hours, 24 hours, 2 days, 7 days, 14 days and 21 days after antibody administration. Blood was collected in a vial containing heparin and centrifuged at 10,000 g for 5 minutes. Plasma was stored at -20°C until the mAb concentration was determined.

Human IgG concentration was determined using a sandwich ELISA. Mouse mAb anti-human IgG-kappa clone MH16 (#M1268, CLB Sanquin, Netherlands) coated on a 96-well micron ELISA plate (Grainer, Germany) at a concentration of 2 μg/mL was used as a capture antibody I did. After the plate was blocked with PBS supplemented with 2% chicken serum, samples were added and serially diluted in ELISA buffer (PBS supplemented with 0.05% Tween 20 and 2% chicken serum) and room temperature for 1 hour on a plate shaker. Incubated at (RT). Plates were subsequently incubated with goat anti-human IgG immunoglobulin (#109-035-098, Jackson, West Grace, PA)) and 2,2'-azino-bis(3-ethylbenztia) Zoline-6-sulfonic acid) (ABTS; Roche, Mannheim, Germany). The absorbance was measured at 405 nm in a microplate reader (Biotech, Winuskey, Vermont).

SCID mice were chosen because they have a low plasma IgG concentration and thus the clearance of IgG is relatively slow. This provides a very sensitive PK model for detecting changes in clearance due to the reduced binding of the Fcγ-moiety to the neonatal Fc receptor (FcRn).

Statistical tests were performed using GraphPad Prism version 4 (GraphPad software).

Figure 12 shows that wild type HuMab-7D8 and IgG1-7D8-E345R both bind well to mouse, human and cynomolgus FcRn. The binding of IgG1-7D8-E345R was somewhat better than wild type 7D8.

13 shows the plasma concentration over time. There was no difference in the change (clearance) of plasma concentration over time of either wild-type HuMab-7D8 versus IgG1-7D8-E345R, -S440K or K322A.

Example 14

Use of Fc-Fc stabilizing mutation E345R for increased bactericidal activity of IgG antibodies against bacteria expressing Fc-binding surface protein

The complement cascade system is an important host defense mechanism against pathogens and can be divided into three different activation pathways that recognize pathogens: i) antibody-mediated classical pathways that are activated upon C1q binding to pathogen-binding antibodies, ii) lectins. , And iii) the complement system directly recognizes the pathogen and is triggered by it in the absence of the antibody. The three pathways converge at the stage of C3 cleavage and C3b deposition. Microorganisms have developed multiple mechanisms of complement avoidance, one of which is mediated by protein A (Joiner Ann. Rev. Microbiol. (1988) 42:201-30; Foster Nat Rev Microbiol (2005) Dec;3( 12):948-58). Protein A was first identified in the cell wall of Staphylococcus aureus, and is well known for its binding to the Fc region of IgG (Deisenhofer et al., Biochem (1981) 20, 2361-70; Uhlen et al. , J. Biol. Chem (1984) 259,1695-1702). So far, the anti-phagocytic effect of protein A and S. Aureus's role in pathogenesis was explained by the interaction between protein A and IgG, which inaccurate the orientation of antibodies recognized by the neutrophil Fc receptor (Foster Nat Rev Microbiol (2005) Dec;3(12). ):948-58).

Example 11 shows that CDC mediated by B cell-specific IgG1 antibody was inhibited by competing Fc-binding peptide DCAWHLGELVWCT. The peptide targets a consensus binding site on an IgG Fc that matches the binding site for Protein A, Protein G and rheumatoid factor (Delano et al., Science 2000 Feb 18;287(5456):1279-83). Based on these data, the Protein A-mediated bacterial complement avoidance mechanism could act by competing for Fc binding, resulting in destabilization of the Fc-Fc interaction of microbial-specific antibodies and consequently inhibition of antibody-mediated complement activation. It is believed to be possible. In addition, Example 11 also shows that B cell-specific IgG1 antibodies containing the CDC-enhancing E345R mutation are less sensitive to inhibition of CDC by competing Fc-binding peptide DCAWHLGELVWCT than wild-type parent antibodies. By extrapolating these results to the Fc-binding protein expressed on the microorganism, the increased stabilization of the IgG1 Fc-Fc interaction by the E345R mutation indicates that microbial-specific antibodies can be used to prevent pathogens through Fc binding competition by microbial surface proteins, such as protein A. It will make you less susceptible to complement inhibition by the escape strategy. Consequently, introduction of the E345R mutation in an IgG antibody that is functional against bacteria will increase C3b deposition on bacteria and increase bactericidal activity compared to the wild-type parent antibody.

As an in vitro measure for complement-mediated bacterial killing, phagocytosis by neutrophils and production of C3a in plasma (which is consistent with C3b deposition on bacteria) can both be determined as described below. Actually, S. C3b deposition on aureus has been demonstrated to enhance phagocytosis and correlate with bacterial killing (Rooijakkers et al., Nature Immunology 2005: 6,920-927).

s. The bacterial culture in which aureus is growing logarithmically will be labeled with FITC by incubating with 100 μg/mL FITC for 1 hour at 37° C. in 0.1 M carbonate buffer (pH 9.6). Human polymorphic nuclear cells (PMN) will be isolated using a Ficoll gradient. FITC-labeled bacteria will be opsonized with a series of concentrations of specific antibodies with or without mutation E345R. Phagocytosis was achieved by incubating ¹ × ^{10 8} opsonized FITC-labeled bacteria with human PMN in the presence of 25% IgG-depleted serum as complement source in a total volume of 200 μL at 37° C. for 25 minutes with vigorous shaking. It will be done in vitro. Cells will be fixed and red blood cells will be lysed by incubation at room temperature for 15 minutes with BD FACS lysis solution. After washing, phagocytosis will be measured by FACS. Neutrophil populations will be selected via forward and side scatter gating, and phagocytosis will be expressed as mean fluorescence in the neutrophil population. Alternatively, C3a production will be measured in the sample by ELISA as a measure for complement activation and C3b deposition.

S. containing the E345R mutation. Aureus-specific antibodies are expected to induce more complement activation and phagocytosis by neutrophils than wild-type parent antibodies. An example of an antibody that can be used in these experiments is the chimeric monoclonal IgG1 pagibaximab (BSYX-A110; Biocinexus) that targets lipoteicosan (LTA) embedded in the cell wall of Staphylococcus ( Baker, Nat Biotechnol. 2006 Dec; 24(12):1491-3; Weisman et al., Int Immunopharmacol. 2009 May; 9(5):639-44).

Example 15

Use of CDC-inhibiting mutations to limit CDC activation to target cells bound simultaneously by a mixture of two different therapeutic monoclonal antibodies

As described in Example 6, CD20 antibody 7D8 mutations K439E and S440K reduced CDC efficacy as monoclonal antibodies. Mixing 7D8 antibodies containing these mutations restored CDC. Thus, efficient CDC was limited to cells bound simultaneously by two mutant antibodies. As described in Example 10, CD38 antibody 005 mutations K439E and S440K reduced CDC efficacy as monoclonal antibodies. Mixing 005 antibodies containing these mutations restored CDC. Thus, efficient CDC was limited to cells bound simultaneously by two mutant antibodies.

It may be advantageous to limit the efficient induction of CDC to target cells expressing two specific antigens simultaneously, using their combined expression to improve the selectivity of CDC induction. To limit CDC induction to cells bound simultaneously by both CD20 and CD38 antibodies, a pair of 7D8-K439E and 005-S440K or a pair of 7D8-S440K and 005-K439E was added separately in the CDC experiment as follows, or 1 :1 will mix. 0.1 x 10 ⁶ Daudi or Raji cells in a round-bottom 96-well plate with a series of concentrations (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg/mL) of crude antibody or antibody mixture. It will be pre-incubated at RT on a shaker for 15 minutes in a total volume of 100 μL. Then 25 μL normal human serum will be added as a source of complement (20% final concentration) and incubated for 45 minutes in a 37° C. incubator. The reaction will be stopped by placing the plate on ice. 10 μL propidium iodide will be added and cell lysis will be determined by FACS. 7D8-K439E, 005-S440K, 7D8-S440K and 005-K439E are expected to exhibit limited CDC efficacy. The simultaneous addition of 7D8-K439E and 005-S440K is expected to restore specifically efficient CDC on cells expressing both CD20 and CD38. Likewise, a mixture of 7D8-S440K and 005-K439E is expected to recover specifically efficient CDC on cells expressing both CD20 and CD38.

Example 16

Increased specificity of CDC enhanced by combining E345R with complementary inhibitory mutations K439E and S440K in a mixture of two different monoclonal antibodies

As described in Example 6, CD20 antibody 7D8 mutations K439E and S440K reduced CDC efficacy as monoclonal antibodies. Mixing 7D8 antibodies containing these mutations restored CDC. Thus, efficient CDC was limited to cells bound simultaneously by two mutant antibodies. As described in Example 10, CD38 antibody 005 mutations K439E and S440K reduced CDC efficacy as monoclonal antibodies. Mixing 005 antibodies containing these mutations restored CDC. Thus, efficient CDC was limited to cells bound simultaneously by both mutant antibodies.

It may be advantageous to limit the enhancement of CDC induction to target cells expressing the two specific antigens simultaneously, using their combined expression to improve the selectivity of enhanced CDC induction. It may also be advantageous to limit the enhancement of CDC induction to target cells that are bound simultaneously by a mixture of at least two different antibodies, wherein the antibodies are on the same cell surface antigen at two different epitopes simultaneously, or at two cross-competitions. Binds at the same, similar, or identical epitope.

Thus, in order to limit the enhanced CDC induction to cells bound simultaneously by both CD20 and CD38 antibodies, the CDC enhancing mutation E345R was replaced with antibodies 7D8-E345R/K439E, 7D8-E345R/S440K, 005-E345R/S440K and 005- Combined with a CDC inhibitory mutation in E345R/K439E. These antibodies were added separately or mixed 1:1 in the CDC experiment as follows. 0.1 x 10 ⁶ Wien133 cells (other cell types such as Daudi or Raji cells can also be used) in a round-bottom 96-well plate in a series of concentrations of crude antibody (7D8-E345R/K439E, 7D8-E345R /S440K, 005-E345R/S440K, or 005-E345R/K439E at a final concentration of 0.056-10,000 ng/mL at 3-fold dilution) or antibody mixture (final concentration mixed with 0-333 ng/mL CD38 antibody at 3-fold dilution 0.01 μg/mL CD20 antibody; or 3.3 μg/mL CD38 antibody mixed with 0.0056-1,000 ng/mL CD20 antibody at 3-fold dilution) and a total volume of 100 μL were pre-incubated at RT on a shaker for 15 minutes. Subsequently, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated for 45 minutes in a 37° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.

A series of concentrations of 005-E345R/K439E or 005-E345R/S440K antibodies were added to a fixed concentration of 0.01 μg/mL 7D8 double mutant antibody (maximum concentration with minimal CDC against Wien133 cells as a single agent as determined from Figure 14A. ) To prepare a complementary combination 005-E345R/K439E + 7D8-E345R/S440K or 005-E345R/S440K + 7D8-E345R/K439E. Figure 14c shows that the 005 double mutant CD38 antibody dose-dependently induced CDC in the presence of a fixed concentration of complementary 7D8-E345R/K439E or 7D8-E345R/S440K CD20 antibodies, respectively. The CDC efficacy by these complementary combinations (FIG. 14C) was comparable to the 005-E345R single mutant (enhancer) antibody as a single agent (FIG. 14B ). In contrast, in the presence of unrelated antibody b12, 005-E345R/K439E and 005-E345R/S440K both showed little or no CDC at the series of concentrations tested (005-E345R/K439E as a single agent shown in FIG. Or equivalent to 005-E345R/S440K).

A series of concentrations of 7D8-E345R/K439E or 7D8-E345R/S440K antibodies were added to a fixed concentration of 3.3 μg/mL 005 double mutant antibody (representing a small but limited CDC against Wien133 cells as a single agent as determined from Figure 14B) and By mixing the complementary combination 7D8-E345R/K439E + 005-E345R/S440K or 7D8-E345R/S440K + 005-E345R/K439E was prepared. Figure 14D shows the CDC in the presence of complementary 005-E345R/K439E or 005-E345R/S440K CD38 antibodies, respectively, even at the lowest concentration tested, in which the 7D8 double mutant CD20 antibody resembles only a few 7D8 double mutant antibody molecules per cell. It shows that it was induced very efficiently. To eliminate the contribution of increased Fc-tail density on the cell membrane to the observed enhanced CDC by a mixture of 7D8 and 005 antibodies with complementary K439E and S440K mutations, antibody combinations with non-complementary mutations were also tested. . 14D shows that the non-complementary combination showed much lower CDC efficacy than the complementary combination as a result of less efficient Fc-Fc interactions than the complementary combination.

These data show that the induction of (enhanced) CDC by the therapeutic antibody in this case binds simultaneously to a mixture of two complementary antibodies with different antigen specificities, requiring co-expression of both antigens, thereby increasing target cell specificity It is suggested that it may be limited.

As can be seen in Figures 14A and 14B, 7D8-E345R/K439E, 005-E345R/S440K, 7D8-E345R/S440K and 005-E345R/K439E showed limited CDC efficiency compared to 7D8-E345R alone. It was further found that a mixture of 7D8-E345R/K439E and 7D8-E345R/S440K as a single agent enables CDC with enhanced efficiency compared to wild type 7D8 antibody. Likewise, it was observed that a mixture of 005-E345R/K439E and 005-E345R/S440K as a single agent enables CDC with enhanced efficiency compared to wild type 005 antibody (data not shown).

Example 17

Use of CDC-inhibitory mutants to limit efficient CDC activation to antibody complexes consisting exclusively of therapeutically administered antibodies

As described in Example 6, the CD20 antibody 7D8 double mutant K439E/S440K restored the reduced CDC efficiency by the K439E or S440K single point mutant. As described in Example 10, the CD38 antibody 005 double mutant K439E/S440K restored CDC efficiency inhibited by the K439E or S440K single point mutant. As observed, single point mutation disrupts the Fc:Fc interaction with unmutated amino acids on opposite sides of the Fc:Fc interface. Introduction of complementary mutations on opposite sides of the Fc:Fc interface restored CDC efficiency. Thus, efficient CDC is clearly limited to antibody complexes consisting exclusively of antibodies containing both mutations.

In another example, the induction of CDC is limited to antibody complexes consisting exclusively of therapeutically administered antibodies. To limit CDC induction to cells bound exclusively by therapeutic CD20 or by CD38 antibody, the CDC inhibitory mutations K439E and S440K will be combined in antibodies 7D8-K439E/S440K or 005-K439E/S440K. These antibodies will be added separately in CDC experiments in the absence or presence of non-target specific IgG as follows. 0.1 x 10 ⁶ Daudi or Raji cells in a round-bottom 96-well plate with a series of concentrations (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg/mL) of crude antibody or antibody mixture. It will be pre-incubated at RT on a shaker for 15 minutes in a total volume of 100 μL. Then 25 μL normal human serum will be added as a source of complement (20% final concentration) and incubated for 45 minutes in a 37° C. incubator. The reaction will be stopped by placing the plate on ice. 10 μL propidium iodide will be added and cell lysis will be determined by FACS.

7D8-K439E/S440K is expected to induce CDC with similar efficiencies to wild type 7D8 antibody. Addition of non-specific IgG to 7D8-K439E/S440K is expected to have no effect on the efficiency of CDC induction for this antibody. Likewise, 005-K439E/S440K is expected to enable CDC with efficiencies similar to wild-type HuMAb 005. Addition of non-specific IgG to 005-K439E/S440K is expected to not affect the efficiency of CDC induction for this antibody.

Example 18

Use of CDC-inhibiting mutations to limit enhanced CDC activation to antibody complexes consisting exclusively of therapeutically administered antibodies

As described in Example 6, the CD20 antibody 7D8 double mutant K439E/S440K restored the reduced CDC efficiency by the K439E or S440K single point mutant. As described in Example 10, the CD38 antibody HuMAb 005 double mutant K439E/S440K restored CDC efficiency inhibited by the K439E or S440K single point mutant. As observed, single point mutation disrupts the Fc:Fc interaction with unmutated amino acids on opposite sides of the Fc:Fc interface. Introduction of complementary mutations on opposite sides of the Fc:Fc interface restored CDC efficiency. Thus, efficient CDC is clearly limited to antibody complexes consisting exclusively of antibodies containing both mutations.

In another example, the enhancement of CDC induction is limited to antibody complexes consisting exclusively of therapeutically administered antibodies. By screening and selection of mutations that stimulate the Fc:Fc interaction used for CDC stimulation, it was possible to identify mutations capable of forming CDC-induced antibody complexes with serum antibodies that are not specific for the antigenic target of interest. . In order to limit the enhanced CDC induction to cells bound exclusively by the complex of CD20 or by the CD38 antibody, the CDC enhancing mutation E345R was added to the CDC inhibitory mutation in antibody 7D8-E345R/K439E/S440K or 005-E345R/K439E/S440K Will be combined with These antibodies will be added separately in CDC experiments in the absence or presence of non-target specific IgG as follows. 0.1 x 10 ⁶ Daudi or Raji cells in a round-bottom 96-well plate with a series of concentrations (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg/mL) of crude antibody or antibody mixture. It will be pre-incubated at RT on a shaker for 15 minutes in a total volume of 100 μL. Then 25 μL normal human serum will be added as a source of complement (20% final concentration) and incubated for 45 minutes in a 37° C. incubator. The reaction will be stopped by placing the plate on ice. 10 μL propidium iodide will be added and cell lysis will be determined by FACS.

7D8-E345R/K439E/S440K is expected to induce CDC with enhanced efficiency compared to wild-type HuMAb 7D8. Addition of non-specific IgG to 7D8-E345R/K439E/S440K is expected to have no effect on the efficiency of CDC induction compared to wild type 7D8 antibody. Likewise, it is expected that 005-E345R/K439E/S440K will enable CDC with enhanced efficiency compared to wild type 005 antibody. The addition of non-specific IgG to 005-E345R/K439E/S440K is expected to have no effect on the efficiency of CDC induction compared to wild-type 005 antibody.

Example 19

Use of a mutant screening approach to identify mutations that stimulate Fc:Fc interaction mediated antibody oligomerization detected by CDC assay

As described in Examples 6 and 10, a CDC-stimulated amino acid mutation was identified on a number of cell lines expressing variable levels of antigen against antibodies recognizing two different target antigens, CD20 and CD38. Surprisingly, the single point mutation E345R proved sufficient to allow CDC-dependent cell lysis of Wien133 cells to be applied to anti-CD38 antibody 005 that failed to lyse these cells by CDC in the wild-type IgG1 format.

Other mutations on or around the Fc:Fc interface could stimulate oligomerization and CDC in a similar fashion. Alternatively, the mutation could indirectly stimulate oligomerization, for example by inducing an Fc:Fc interaction in an allosteric manner.

To determine if other amino acid mutations can stimulate Fc-mediated antibody oligomerization, a library of anti-CD38 IgG1-005 mutants was screened both separately using the CDC assay, e.g. Fc:Fc interface Mixed in a pairwise fashion to select pairs of amino acids that interact across. However, the same strategy can be applied to other antibodies, such as another IgG1 or IgG3 antibody.

A library focused on mutations at the positions indicated in Table 16 was generated. Mutations were introduced into the IgG1-005 Fc region using a quick change site-directed mutagenesis kit (Stratagene, USA). Briefly, for each desired mutation site, the full length plasmid DNA template of the 005 heavy chain with the IgG1m(f) allotype was cloned using forward and reverse primers encoding the degenerate codon at the desired position. The resulting DNA mixture was digested using DpnI to remove the source plasmid DNA, and E. It was used to transform coli. The resulting colonies were collected and cultured, plasmid DNA was isolated from these pools, and E. Retransformation into E. coli yielded clone colonies. Mutant plasmid DNA isolated from the resulting colonies was examined by DNA sequencing (LGC genomics, Berlin, Germany). The expression cassette was amplified from plasmid DNA by PCR, and a DNA mixture containing both the mutant heavy and wild type light chains of IgG1-005 was essentially freestyle using 293 pectin (Invitrogen, USA) as described by the manufacturer. HEK293F cells (Invitrogen, USA) were transiently transfected. The supernatant of the transfected cells containing the antibody mutant was collected. Mutant antibody supernatants were screened in CDC assay, both individually and in pairwise mixtures as follows.

0.1 x 10 ⁶ Daudi or Wien-133 cells (other cell types such as Raji cells may be used) in round-bottom 96-well plates for 15 min with 1.0 ug/ml of crude antibody and a total volume of 100 μL During the pre-incubation at RT on a shaker. Then, 30 μL normal human serum was added as a source of complement (30% final concentration) and incubated for 45 minutes in a 37° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.

Oligomerization as detected by CDC efficiency when the mutations listed in Tables 16, 17 and 18 are as single mutants or when mixed with other mutants, for example, when opposed to mutations across the Fc:Fc interface. It was chosen for his ability to enhance. Optionally, mutations can be further screened for their ability to not impair FcRn, Protein-A or Protein-G binding, ADCC, ADCP or other effector functions mediated by the Fc domain. Combining these stimulating point mutations into one Fc domain can further stimulate oligomerization and CDC efficiency.

Mutations in the CH2-CH3 region contained within CD38 antibody 005 were tested for their ability to inhibit oligomerization as determined by CDC on Daudi cells. The lysis of the mutant antibody was compared to wild type 005, and the lysis for it was set to 100%. The cutoff for inhibition was set to ≤66% dissolution. When measured in this way, most of the tested mutations inhibited CDC (see Table 16).

Mutations in the CH2-CH3 region incorporated in CD38 antibody 005 were tested for their ability to enhance oligomerization as determined by CDC on Wien133 cells (Table 17). Wild type CD38 antibody 005 is unable to induce CDC on Wien133 cells. Mutants showing ≧39% cell lysis were scored as enhancement. Completely unexpectedly, substantially all of the obtained substitutions of amino acids E345 and E430 stimulated cell lysis by CDC. To demonstrate these results, amino acids E345, E430 and S440 were replaced with each possible mutation by site-directed mutagenesis, and a new human serum batch was used to enhance oligomerization as determined by CDC for Wien133 cells. Tested for ability and obtained somewhat more efficient dissolution (Table 18). Again, all substitutions of E345 and E430 led to efficient CDC of Wien133 cells.

The following preferred mutations caused ≥39% cell lysis of Wien133 cells: P247G, I253V, S254L, Q311L, Q311W, E345A, E345C, E345D, E345F, E345G, E345H, E345I, E345K, E345L, E345M, E345N, E345P, E345Q, E345R, E345S, E345T, E345V, E345W, E345Y, D/E356G, D/E356R, T359R, E382L, E382V, Q386K, E430A, E430C, E430D, E430F, E430G, E430H, E430I, E430L, E430M, E430N E430P, E430Q, E430R, E430S, E430T, E430V, E430W, E430Y, Y436I, S440Y and S440W.

[Table 16]

[Table 17]

[Table 18]

Example 20

In vivo efficacy of IgG1-7D8-E345R in a subcutaneous B-cell lymphoma xenograft model

The in vivo anti-tumor efficacy of the IgG1-7D8-E345R antibody was evaluated in a subcutaneous model using Raji-luc #2D1 cells. These cells show -300,000 CD20 molecules per cell (determined by QIFIKIT analysis, data not shown) and high complement defense receptor expression. Cells were treated with 10% cosmic calf serum (Hyclone, Logan, Utah), penicillin and streptomycin, 1% (v/v) sodium pyruvate and 1 μg/mL puromycin (P-8833, Sigma, Zvindrecht). It was cultured in RPMI containing. Cells were harvested in log-phase (approximately 70% confluent growth rate). 6-11 week old female SCID mice (CB-17/IcrPrkdc-scid/CRL) were used (Charles-River). On day 0, 5×10 ⁶ Raji-luc #2D1 cells in 200 μL PBS were injected subcutaneously into the right flank of each mouse. Tumor development was monitored by caliper measurement. When the mean tumor volume was 100 mm ³ (about day 7), mice were divided into groups (n = 9) and by intraperitoneal (ip) injection of a single dose (2.5 mg/kg) of 50 μg antibody per mouse. Processed. All antibody samples were supplemented with unrelated antibody b12 to obtain a total antibody concentration of 0.5 mg/mL. Treatment groups are shown in Table 18. After 7 days of treatment, blood samples were obtained to determine human IgG serum levels to review correct antibody administration. Tumors were measured at least twice a week using a caliper (PLEXX) to an endpoint tumor volume of 1500 mm ³ , when the tumor showed ulceration or until severe clinical signs were observed.

[Table 18]

15A shows average tumor growth on day 22 when all groups are still complete. Wild type antibody IgG1-7D8 somewhat inhibited tumor growth compared to negative control antibody IgG1-b12, but this was not statistically significant. Only IgG1-7D8-E345R significantly inhibited tumor growth compared to the negative control antibody IgG1-b12 (one-way ANOVA analysis p<0.01).

15B shows a Kaplan-Meier plot of the percentage of mice with tumor size smaller than 700 mm ³ . Compared to mice treated with the negative control antibody IgG1-b12, tumor formation was significantly delayed in mice treated with the IgG1-7D8-E345R antibody (Mantel-Cox assay p<0.01), but wild-type IgG1- This was not the case in mice treated with 7D8.

These data show that the E345R mutation enhanced the in vivo anti-tumor efficacy of CD20 antibody 7D8.

Example 21

In vivo efficacy of IgG1-005-E345R in subcutaneous B cell lymphoma model

The in vivo anti-tumor efficacy of the IgG1-005-E345R antibody was evaluated in a subcutaneous model using Raji-luc #2D1 cells. These cells show ~150,000 CD38 molecules per cell (determined by QIFIKIT analysis, data not shown) and high complement defense receptor expression. The protocol for tumor inoculation and measurement is basically the same as described in Example 20. On day 0, 5×10 ⁶ Raji-luc #2D1 cells in 200 μL PBS were sc injected into the right flank of SCID mice. When the average tumor volume was 100 mm ³ (about day 7), mice were classified into groups (n = 7) and treated by ip injection of a single dose (25 mg/kg) of 500 μg antibody per mouse. Treatment groups are shown in Table 19. Tumors were measured to an endpoint tumor volume of 1500 mm ³ or until the tumor showed ulceration or severe clinical signs were observed to avoid major discomfort.

16A shows average tumor growth on day 21 when all groups were still complete. The wild-type antibody IgG1-005 somewhat inhibited tumor growth, but this was not statistically significant. Only IgG1-005-E345R significantly inhibited tumor growth on day 21 compared to the unrelated antibody control group (one-way ANOVA p<0.05).

16B shows a Kaplan-Meier plot of the percentage of mice with tumor sizes smaller than 500 mm ³ . Tumor formation was significantly delayed in mice treated with IgG1-005-E345R antibody compared to mice treated with negative control antibody IgG1-b12 (Mandel-Cox assay p<0.001) or wild-type IgG1-005 (p<0.05). .

These data show that the introduction of the E345R mutation in CD38 antibody 005 enhanced anti-tumor activity in vivo.

[Table 19]

Example 22

Monovalent target binding further enhances CDC efficacy of E345R antibody

The molecular surface of the IgG1 hexameric ring observed in the b12 crystal structure is for each IgG in the hexameric ring, one of the two C1q binding sites facing upward of the ring structure and the other facing downward, and also each One Fab-arm of the antibody of is shown to be oriented upward and one oriented downward, allowing only one Fab-arm per antibody to participate in antigen binding, which allows monovalent binding per antibody molecule within the hexameric antibody ring. Suggest. Monovalent is capable of antigen binding of antibodies in a hexamerization compatible orientation. To test this hypothesis, the CDC efficacy of the bispecific CD38/EGFR antibody with the E345R mutation was tested in CD38-positive, EGFR-negative Wien133 cells (this bispecific antibody can only monovalently bind to it via CD38. ) And also compared to the CDC efficacy of the bivalent binding CD38 antibody with the E345R mutation. The human monoclonal antibody HuMax-EGFr (2F8, described in WO 2004/056847) was used as a reference for the EGFR antibody described in this example.

Bispecific antibodies were generated in vitro according to the Duobody™ platform, ie according to 2-MEA-induced Fab-arm exchange as described in WO 2011/147986. The basis for this method is the use of complementary CH3 domains, which promote heterodimer formation under specific assay conditions. In order to enable the production of bispecific antibodies by this method, an IgG1 molecule was created that carries a specific mutation in the CH3 domain: one of the parental IgG1 antibodies carries the F405L mutation and the other parental IgG1 antibody the K409R mutation. . To generate bispecific antibodies, these two parent antibodies (for each antibody, at a final concentration of 0.5 mg/mL) were added to 100 μL TE with 25 mM 2-mercaptoethylamine-HCl (2-MEA). Incubated at 37° C. for 90 minutes with a total volume of. The reduction reaction is stopped when the reducing agent 2-MEA is removed using a rotating column (Microcon centrifugal filter, 30k, Millipore) according to the manufacturer's protocol.

For CDC assay, 0.1 x 10 ⁶ Wien133 cells were prepared in a round-bottom 96-well plate with a series of concentrations of antibodies (0.01 to 10.0 μg/mL) in a total volume of 100 μL at RT on a shaker for 15 min. -Incubated. Subsequently, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated for 45 minutes in a 37° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.

Figure 17 shows that CD38 antibody without E345R mutation (wild type IgG1-005 and IgG-b12-K409R x IgG1-005-F405L) did not induce death of Wien133 cells, as expected. In addition, the EGFR antibody IgG1-2F8-E345R/F405L, which does not bind to EGFR-negative Wien133 cells (data not shown), did not induce CDC as expected. Introduction of the K409R mutation did not affect the ability of the IgG1-005-E345R antibody to induce -60% death on Wien133 cells (described in Example 10). Interestingly, the bispecific CD38/EGFR antibody IgG1-005-E345R/K409R x IgG1-2F8-E345R/F405L, which can only monovalently bind to CD38-positive, EGFR-negative Wien133 cells, resulted in increased maximal CDC killing (~ 60% to -100% death).

These data show that monovalent targeting can further enhance the maximum killing capacity of antibodies containing the CDC enhancing E345R mutation. In addition, these data show that the E345R oligomerization enhancing mutation can be applied to other antibody types, such as Duobodies, as measured by enhancing CDC activity.

Example 23

Oligomerization enhancing E345R mutation can be applied to other antibody formats such as Duobody™

The effect of the E345R mutation was tested in a duobody format bispecific antibody. CDC assays were performed using CD20/CD38 bispecific antibodies on CD20-positive, CD38-positive Wien133 and Raji cells.

Bispecific antibodies were generated as described in Example 22. For CDC assay, 0.1 x 10 ⁶ Wien133 or Raji cells were placed in a round-bottom 96-well plate with a series of concentrations (0.01-30.0 μg/ml) of antibody and a total volume of 100 μL at RT on a shaker for 15 min. Pre-incubated. Subsequently, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated for 45 minutes in a 37° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.

Figure 18 shows that the introduction of the E345R mutation enhanced the CDC of the bispecific IgG1-005-F405L x IgG1-7D8-K409R antibody on Wien 133 (Figure 18A) and Raji (Figure 18B) cells. These data show that E345R oligomerization enhancing mutations can be applied to other antibody formats to enhance CDC activity.

Example 24

E345R restores CDC by EGFR antibody 2F8, which can be further enhanced by monovalent target binding

As described in Examples 6, 10 and 26, E345R enhanced or restored CDC against antibodies recognizing different blood tumor targets (CD20 and CD38). To extend the assay to solid tumor antigens, the effect of E345R on the CDC ability of EGFR antibody 2F8 was tested on A431 epidermal carcinoma cells. In addition, the effect of monovalent EGFR targeting on E345R-mediated CDC induction was evaluated using a bispecific EGFRxCD20 antibody (IgG1-2F8-E345R/F405L x IgG1-7D8-E345R/K409R) on EGFR-positive, CD20-negative A431 cells. And tested.

Bispecific antibodies were generated as described in Example 22. For the CDC assay, 5 x 10 ⁶ A431 cells/mL were labeled with 100 μCi ⁵¹ Cr for 1 hour at 37°C. The cells were washed 3 times with PBS and resuspended in the medium at a concentration of 1 x 10 ⁵ cells/mL. 25,000 labeled cells were incubated for 15 min at RT in a round-bottom 96-well plate with a series of concentrations (0-30 μg/mL in 3-fold dilution) of crude antibody and a total volume of 100 μL. Subsequently, 50 μL normal human serum dilution was added as a source of complement (25% final concentration) and incubated for 1 hour in a 37° C. incubator. Cells were spun down (3 min at 300xg), and 25 μL supernatant was added to 100 μL Microscint in a white 96 well Optiplate (PerkinElmer) while incubating on a shaker (750 rpm) for 15 minutes. ⁵¹ Cr release was determined in counts per minute (cpm) on a scintillation counter. Maximum lysis (100%) was determined by the ⁵¹ Cr level measured in the supernatant of Triton X-100-treated cells. Spontaneous lysis was determined by the ⁵¹ Cr level measured in the supernatant of cells incubated without antibody. Specific cell lysis was calculated according to the following formula: specific lysis = 100 x (cpm sample-cpm spontaneous lysis) / (cpm maximum-cpm spontaneous lysis).

19 shows that IgG1-2F8-E345R/F405L can lyse A431 cells by CDC, but wild-type 2F8 cannot kill A431 cells. These data show that CDC activity can be restored in EGFR antibody 2F8 by introduction of the E345R mutation. This potentially extends the applicability of the CDC enhancing E345R mutation to antibodies targeting solid tumor antigens.

The bispecific EGFRxCD20 antibody IgG-2F8-E345R/F405L x IgG1-7D8-E345R/K409R showed additional CDC enhancement on EGFR-positive, CD20-negative A431 cells.

These data further support the hypothesis that monovalent facilitates the formation of the hypothesized Fc-Fc interaction and subsequent CDC induction for the CD38 binding antibody described in Example 22.

Example 25

E345R enhances or restores CDC by CD38 antibody 003 and CD20 antibody 11B8 and rituximab

As described in Examples 6, 10 and 24, E345R enhances or induces the CDC activity of several antibodies with different target specificities (CD20, CD38 and EGFR) when tested on multiple cell lines expressing variable levels of antigen. . Therefore, the introduction of the E345R mutation has come to be considered as a general mechanism for enhancing or restoring CDC against existing antibodies. To further support this, the effect of the E345R mutation on CDC was tested against more antibodies with variable intrinsic CDC efficacy against Daudi and Wien133 cells: CD38 antibody 003 described in WO 2006/099875 and WO 2005/103081. CD20 antibodies rituximab (type I) and 11B8 (type II) described. CD20 antibodies can be separated into two subgroups (Beers et al. Seminars in Hematology 47, (2) 2010, 107-114). Type I CD20 antibodies show a remarkable ability to activate complement and elicit CDCs by redistributing CD20 molecules within the plasma membrane into lipid rafts that cluster the antibody Fc regions and enabling improved Clq binding. Type II CD20 antibodies do not significantly change the CD20 distribution and, without concomitant clustering, are relatively ineffective in CDC.

0.1 x 10 ⁶ Daudi or Raji cells in a round-bottom 96-well plate with 70 μL of crude antibody at serial concentrations (0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0 μg/mL). Pre-incubate at RT on a shaker for 15 minutes with a total volume of. Then, 30 μL normal human serum was added as a source of C1q (30% final concentration) and incubated for 45 minutes in a 37° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.

20 shows that the E345R mutation enhanced CDC for all treated antibodies in both (a) Daudi cells and (b) Wien133 cells. Interestingly, all antibodies that do not induce CDC in the wild-type format at the concentrations used have the E345R mutation: CD38 mAb 003 and CD20 type II mAb 11B8 in Daudi cells, and of CD38 mAb 005 and 003 and CD20 type II mAb 11B8 in Wien133 cells. After introduction, CDC was efficiently induced. These data more specifically suggest that the enhancement of antibody oligomerization by the introduction of the E345R mutation is a general mechanism for enhancing or restoring CDC by existing antibodies.

Example 26

E345R promotes internalization of tissue factor antibodies

To test whether enhanced oligomerization could induce increased antibody internalization, colocalization studies of the lysosome marker LAMP1 and wild type and E345R mutated tissue factor (TF) antibodies were performed by confocal microscopy.

SK-OV-3 cells were grown for 1 day at 37° C. in standard tissue culture medium on glass coverslips (1.5 microns thick, Thermo Fisher Scientific, Braunschweig, Germany). Cells were grown for 1 hour During pre-incubation with 50 μg/mL leupeptin (Sigma) to block lysosome activity, and then 10 μg/mL tissue factor (TF) antibody (WO 2010/066803) was added. Alternatively, the cells were incubated for 16 hours at 37° C. The cells were then washed with PBS and incubated with 4% formaldehyde (Klinipath) for 30 minutes at room temperature (RT), The slides were incubated with blocking buffer (0.1). % Saponin (Roche) and PBS supplemented with 2% BSA (Roche)) and incubated with blocking buffer containing 20 mM NH ₄ Cl for 20 minutes to quench formaldehyde The slides were again quenched with blocking buffer. Wash and goat-anti-human IgG-FITC (Jackson) to identify mouse-anti-human CD107a-APC (BD Pharmingen) and TF antibodies to identify lysosome LAMP1 at RT for 45 min. The slides were washed again with blocking buffer and 20 μL mounting medium (6 grams glycerol (Sigma) and 2.4 grams Mowiol 4-88 (Omnilabo)) were added to 12 mL 0.2M Tris (Sigma). ) After adding pH 8.5, dissolve in 6 mL distilled water incubated at 50-60° C. for 10 minutes; aliquot the mounting medium and store at -20° C. Mounted on a microscope slide overnight using a slide 63×1.32- Imaged using a Leica SPE-II confocal microscope (Leica Microsystems) equipped with a 0.6 oil immersion objective lens and LAS-AF software.

Co-localization of 12-bit grayscale TIFF images using MetaMorph® software (version Meta Series 6.1, Molecular Devices Inc, Sunnyvale, CA, USA) Was analyzed. Images were stacked and the background was subtracted. The same threshold setting was used for all FITC images and all APC images (set manually). The colocalization was expressed as the pixel intensity of FITC within the region of interest (ROI), and the ROI consists of all APC positive regions. To compare different slides stained with different TF antibodies, images were normalized using the pixel intensity of APC. The lysosome marker LAMP1 (CD107a) was stained using mouse-anti-human CD107a-APC. The pixel intensity of LAMP1 should not differ between the various TF antibodies imaged.

The normalized values for co-localization of FITC and APC are expressed as arbitrary units according to the formula [(TPI FITC x co-localization percentage)/100] x [1/TPI APC].

Co-localization Percentage = TPI FITC / TPI APC co-localized with APC pixels

TPI, total pixel intensity

Figure 21 depicts the amount of FITC pixel intensity of wild-type and E345R mutated TF antibodies overlapping with APC-labeled lysosome markers. For each antibody or condition tested, three different images were analyzed from one slide containing -1, 3 or >5 cells. Variations were observed between different images within each slide. Still, it was clear that the E345R mutations for antibodies 011 and 098 resulted in increased lysosome colocalization after 1 hour incubation compared to wild type 011 and 098. These results indicate that mutant E345R can induce more rapid internalization and lysosome colocalization and thus enhance antibody drug conjugates.

Example 27

CDC enhanced by the E345R mutation in Rituximab in different B cell lines with similar CD20 expression but with different levels of membrane-bound complement regulatory protein

Examples 25 and 28 show that the CDC efficacy of wild-type rituximab against Daudi and Wien133 cells was enhanced by introducing the E345R mutation. This enhanced CDC efficacy results from E345R-mediated stabilization of the Fc-Fc interaction. Subsequently, the hexameric antibody ring structure co-formed on the target cell membrane can facilitate the capture and concentration of the activated complement component close to the cell membrane, thereby facilitating the efficient generation of membrane attack complexes. As a result of this efficient complement activation, the inhibitory effect of the membrane-bound complement regulatory protein (mCRP) can be partially overcome. Overexpression of mCRP, such as CD55, CD46 and CD59, is considered a barrier to successful immunotherapy using monoclonal anti-tumor antibodies (Jurianz et al., Mol Immunol 1999 36:929-39; Fishelson et al. Mol Immunol 2003 40:109-23, Gorter et al., Immunol Today 1999 20:576-82, Zell et al., Clin Exp Immunol. 2007 Dec 150(3):576-84). Thus, the efficacy of rituximab-E345R was compared to wild-type rituximab on a series of B cell lines with different levels of mCRP CD46, CD55 and CD59 but with comparable levels of CD20 target expression.

B cell lines Daudi, WIL2-S, WSU-NHL, MEC-2 and ARH-77 are able to express equivalent amounts of CD20 molecules (~250,000 specific antibody-binding capacity-sABC) as determined by QIFIKIT analysis. Yes (data not shown). In order to compare the expression levels of complement regulatory proteins between these cell lines, QIFIKIT analysis was performed to obtain CD46 (mouse anti-human CD46, CBL488, clone J4.48 Chemicon), CD55 (mouse anti-human CD55, CBL511. , Clone BRIC216, Chemicon), and CD59 (mouse anti-human CD59, MCA1054x, clone MEM-43, Sertec).

For the CDC assay, 0.1 x 10 ⁶ cells were placed in a round-bottom 96-well plate with a series of concentrations (0.002-40.0 μg/mL in 4-fold dilution) of saturated antibody and a total volume of 100 μL on a shaker for 15 min. Pre-incubated at RT. Subsequently, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated for 45 minutes in a 37° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS. Maximum CDC-mediated killing was calculated from two independent experiments using the top best-fit values of non-linear fit in GraphPad Prism 5.

22A-D show that introduction of E345R in wild-type rituximab resulted in enhanced CDC efficacy as observed with increased maximal lysis and decreased EC ₅₀ for all tested B cell lines.

22E shows that the maximal CDC-mediated killing induced by the rituximab-E345R mutant is always higher than the wild-type rituximab regardless of the expression level of the membrane-bound complement regulatory protein. These data indicate that the introduction of E345R enhances the therapeutic potential of monoclonal antibodies as tumor cells are less effective in avoiding antibody-mediated complement attack by E345R containing antibodies.

Example 28

Comparison of CDC kinetics for wild type and E345R antibodies

Introduction of the Fc:Fc interaction stabilizing E345R mutation was performed in Example 6 (CD20 antibody 7D8 in Daudi and Raji), Example 10 (CD38 antibody 005 in Daudi, Raji and Wien133) and Example 25 (CD38 antibody 003 in Daudi and Wien133). And CD20 antibodies Rituximab and 11B8) on the different cell lines described in the CDC as observed by decreased EC ₅₀ values and increased maximal lysis for different antibodies. Then, the kinetics of the CDC response were analyzed to further elucidate the difference in CDC efficacy between wild-type and E345R antibodies.

0.1 x 10 ⁶ Raji cells were pre-incubated in round-bottom 96-well plates with antibodies at a saturation concentration (10.0 μg/mL) in a total volume of 100 μL for 15 minutes on a shaker at RT. Then 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for different periods between 0 and 60 minutes. The reaction was stopped by placing the plate on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.

23A shows that the wild-type CD20 antibody IgG1-7D8 showed a maximal CDC-mediated killing of 80% of Raji cells, which was already achieved after 5 minutes under the conditions tested. However, for IgG-7D8-E345R, 80% death of Raji cells was observed much faster after 3 minutes. Maximum dissolution (95%) by IgG-7D8-E345R was also achieved after 5 minutes.

Figure 23B also shows that for the wild-type CD20 antibody Rituximab, which is less potent than 7D8 to induce CDC on the Raji cells used, the introduction of the E345R mutation resulted in faster killing of target cells. Wild-type Rituximab showed a maximum CDC-mediated kill of 32%, which was achieved after 20 minutes. Rituximab-E345R already reached 32% killing after approximately 3 minutes, in particular, maximal dissolution (85%) with Rituximab-E345R was also achieved after 20 minutes.

23C+D shows that used Raji cells resistant to CDC-mediated killing by wild type CD38 antibodies IgG1-003 and IgG1-005 could be killed rapidly by introducing the E345R mutation. IgG1-003-E345R and IgG1-005-E345R already showed maximum CDC (50% and 60%, respectively) after 5 minutes.

In summary, the E345R antibody is more potent than its wild-type counterpart, resulting from the combination of greater potency (lower EC ₅₀ ), increased maximal lysis and faster kinetics of the CDC response.

Example 29

Comparison of CDC kinetics for bispecific antibodies with or without E345R mutation

In Example 23, the E345R mutation can be applied to the CD38xCD20 bispecific antibody IgG1-005-F405L x IgG1-7D8-K409R generated by the Duobody platform, resulting in a reduced EC ₅₀ in the CDC assay for Raji and Wien133 cells. It is described that causes the enhanced killing ability as observed by. The kinetics of the CDC response were then analyzed to further elucidate the difference in CDC efficacy between the CD38xCD20 bispecific antibodies with and without E345R.

0.1 x 10 ⁶ Raji cells were pre-incubated in round-bottom 96-well plates with antibodies at a saturation concentration (10.0 μg/mL) in a total volume of 100 μL for 15 minutes on a shaker at RT. Then 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for different periods between 0 and 60 minutes. The reaction was stopped by placing the plate on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.

Figure 24 shows that the bispecific antibody IgG1-005-F405L x IgG1-7D8-K409R induced a maximal CDC-mediated killing of 83%, which was achieved after 10 minutes. Introduction of E345R resulted in increased maximal killing by IgG1-005-E345R-F405L x IgG1-7D8-E345R-K409R (98%), which was already achieved after 2 minutes. These data indicate that introduction of the Fc-Fc stabilized E345R mutation in the bispecific antibody accelerates CDC-mediated killing of target cells.

Example 30

Comparison of CDC kinetics for monovalent binding antibodies with and without E345R

Example 22 shows that monovalent target binding further enhanced the CDC efficacy of the E345R antibody as observed by increased maximal lysis using the CD38xEGFR bispecific antibody on CD38-positive, EGFR-negative Wien133 cells. Then, the kinetics of the CDC response were analyzed to further account for the difference in CDC-mediated killing ability between monovalent binding antibodies with and without E345R.

Bispecific CD38xEGFR and CD20xEGFR antibodies with or without the E345R mutation were generated in vitro according to the Duobody platform as described in Example 22. The CDC efficacy of the CD38xEGFR bispecific antibody was tested on CD38-positive, EGFR-negative Raji cells, in which the bispecific antibody can only bind monovalently through CD38. 0.1 x 10 ⁶ Raji cells were pre-incubated in round-bottom 96-well plates with antibodies at a saturation concentration (10.0 μg/mL) in a total volume of 100 μL for 15 minutes on a shaker at RT. Then 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for different periods between 0 and 60 minutes. The reaction was stopped by placing the plate on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.

Figure 25 shows that the bispecific antibody CD38xEGFR (IgG1-005-K409R x IgG1-2F8-F405L) induced a maximum CDC-mediated kill of 55%, which was achieved after approximately 10 minutes. Introduction of E345R resulted in an increased maximum kill (96%), which was already achieved within 5 minutes.

Figure 25 shows that the bispecific antibody CD20xEGFR (IgG1-7D8-K409R x IgG1-2F8-F405L) induced a maximal CDC-mediated killing of 85%, which was achieved after approximately 5 minutes. However, when using the CD20xEGFR antibody with E345R introduced, 85% lysis was observed more rapidly after 2 minutes. Maximum lysis (97%) with the E345R CD20xEGFR antibody was also achieved after 5 minutes.

In summary, the introduction of the E345R mutation in these monovalent binding antibodies resulted in more potent antibodies, resulting from the combination of increased maximal lysis and faster kinetics of the CDC response.

Example 31

CDC by combination of therapeutic antibody and E345R/Q386K antibody

As described in Example 19, a mutant CD38 antibody derived from IgG1-005 can induce efficient CDC on Wien133 cells when the E345 position of the wild-type antibody is substituted with any amino acid other than glutamate (E). This suggests that oligomerization, a prerequisite for CDC, is hampered by the presence of the glutamate side chain at position 345 of the antibody. Since E345 on one Fc is in close proximity to Q386 on the opposite second Fc moiety within the hexameric antibody ring structure, E345-mediated interference of oligomerization in the first antibody is possibly at the Q386 position of the second antibody. It can be removed by substitution. This will then allow E345 in the first antibody to better interact with the mutated 386 position in the second antibody when the two antibodies are combined. To test this hypothesis, a CDC assay was performed on Wien133, where wild-type antibodies (IgG1-003, IgG1-005 or IgG1-11B8) were used as an example IgG1-005-E345R/Q386K or IgG1-005-E345R/Q386K /E430G and mixed.

0.1 x 10 ⁶ Wien133 cells were placed in a round-bottom 96-well plate in a series of concentrations (0.0001-20.0 μg/mL at 3.33 fold dilution) of crude IgG1-005-E345R/Q386K, IgG1-005-E345R/Q386K. Pre-incubated at RT on a shaker for 15 minutes in a total volume of 100 μL with or without 1.0 or 10.0 μg/mL wild type IgG1-003, IgG1-005 or IgG1-11B8 antibody with /E430G or control antibody. Subsequently, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated for 45 minutes in a 37° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.

Figure 26a/b/c shows that the CD38 antibody IgG1-005-E345R/Q386K induced CDC-mediated lysis of Wien133 cells in a dose-dependent manner (dashed line). Combining IgG1-005-E345R/Q386K with 1 or 10 μg/mL wild-type CD38 antibody IgG1-003 (Figure 26A) or wild-type CD20 antibody IgG1-11B8 (Figure 26B) resulted in increased maximal cell lysis. Combining IgG1-005-E345R/Q386K with wild-type IgG1-005 inhibited CDC in a dose-dependent manner, possibly by competing for the binding site (FIG. 26C ).

Figure 26d/e/f shows similar results as for the CD38 antibody IgG1-005-E345R/Q386K/E430G.

These data indicate that wild type antibodies IgG1-003 and IgG1-11B8 participate in antibody oligomerization and CDC activation when combined with IgG1-005-E345R/Q386K or IgG1-005-E345R/Q386K/E430G. In this combination, interference with oligomerization by the E345-position present in the wild-type antibody can be eliminated, at least in part, by a Q386K substitution in the mutant antibody. This application is of particular interest to improve therapy with antibodies that are wild-type at the E345 position, such as rituximab, ofatumumab, daratumumab or trastuzumab. In addition, such oligomerization-inducing antibodies can promote the formation of cell-bound complexes with patient-own antibodies that are directed against target cells such as tumor cells or bacteria.

Example 19 describes a number of amino acids other than E345, e.g. E430 and S440, which enhance CDC upon mutation, wherein specific mutations induce efficient CDC in Wien133 cells when introduced to the CD38 antibody IgG1-005 I did. With the exception of the I253 and Y436 mutants, the identified oligomerization-enhancing mutations contact an unmutated amino acid on the opposite second Fc moiety in the hexameric ring structure. Thus, identified oligomerization-enhancing mutations (both alone or in combination) can also be expected to promote oligomerization with unmutated antibodies, and further optimization of these mutants was applied in Example 19. Can be achieved by a selection strategy similar to that of that.

Example 32

E345R induced CDC in IgG2, IgG3 and IgG4 antibody isotypes

To test whether the introduction of oligomerization-promoting mutations can stimulate the CDC activity of non-IgG1 antibody isotypes, isoforms of CD38 antibodies with constant domains of human IgG2, IgG3 or IgG4 by methods known in the art Type variant IgG1-005 was generated to obtain IgG2-005, IgG3-005 and IgG4-005. In addition, oligomerization enhancing E345R mutations were introduced into all of these antibodies to obtain IgG2-005-E345R, IgG3-005-E345R and IgG4-005-E345R. In a similar manner, IgG2-003 and IgG2-003-E345R were also generated from the CD38 antibody IgG1-003. The CDC efficacy of the different isotypes was compared in an in vitro CDC assay.

0.1 x 10 ⁶ Wien133 cells were pre-incubated in round-bottom 96-well plates with 10 μg/mL crude antibody in a total volume of 100 μL for 15 minutes on a shaker at RT. IgG1-005-E345R was added at 3.0 μg/mL. Subsequently, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated for 45 minutes in a 37° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.

Figure 27 shows that IgG2-005, IgG2-003, IgG3-005 and IgG4-005 were unable to efficiently lyse (a) Daudi or (b) Wien133 cells under the tested conditions (observed -20% lysis Is considered background). Introduction of the E345R mutation enabled potent CDC on Daudi cells by all IgG isotypes tested. Although IgG3-005-E345R showed limited CDC activity compared to other isotype variants, these results were confirmed using CDC against Wien133 cells. These data indicate that, in addition to IgG1, oligomerization enhancing mutations such as E345R can also be applied to promote CDC activity of IgG2, IgG3 and IgG4 antibodies.

Example 33

CDC by IgG1-005 and IgG1-005-E345R in an ex vivo CDC assay for patient-derived CD38-positive B cell chronic lymphocytic leukemia (CLL) cells.

Cryopreserved primary cells from CLL patient samples were collected from CDB-IDIBAPS-Hospital Clinic (Dr. Elias Campo), Institute d'Investigacions Biomediques August Pi i, University of Barcelona, Barcelona, Spain. Sunyer, Hospital Clinic, Department of Hematology, Department of Pathology), or from the National Heart, Lung, and Blood Institute (NHLBI)) (Dr. Adrian Wistner (Dr. Adrian Wiestner), National Institutes of Health (NIH) Hematology Pathology Division, Bethesda, NHLBI). Prior consent was obtained from all patients in accordance with the Institutional Ethics Committee of the Hospital Clinic (Barcelona, Spain) or the Institutional Review Board of the NIH and the Declaration of Helsinki. All samples were characterized genetically and by immunophenotype.

CLL samples were divided into two groups according to their CD38 expression as determined by FACS: 5 samples were included in the CD38 high group (50% to 98% of CD38 expression on Daudi cells), 4 samples were CD38 It was included in the lower group (0.5% to 3% of CD38 expression on Daudi cells).

Fluorescently labeled CLL cells (labeled with 5 μM calcein AM) were incubated with antibodies at a series concentration (0.01-10 μg/mL at 10-fold dilution). Normal human serum was then added to antibody-opsonized cells (100,000 cells/well) as a source of complement (10% final concentration) and incubated at 37° C. for 45 minutes. The supernatant was recovered and fluorescence was read on a Synergy™ HT fluorometer as a measure for cell lysis. Cell death was calculated as follows:

Specific lysis = 100 x (sample-spontaneous lysis)/(maximum lysis-spontaneous lysis), where the maximal lysis is determined by the sample of cells treated with 1% Triton, and spontaneous lysis causes the cells to be in the absence of antibodies and Determined from the sample when incubated in the presence.

Figure 28 shows that IgG1-005-E345R strongly enhanced CDC efficacy in both CLL primary cells with high CD38 expression and CLL primary cells with low CD38 expression compared to wild-type IgG1-005.

Example 34

FcRn binding of IgG1-005 mutants compared to wild-type IgG1-005

The neonatal Fc receptor (FcRn) is responsible for the long plasma half-life of IgG by protecting it from degradation. After internalization of the antibody, FcRn binds to the antibody Fc region in the endosome, where the interaction is stable in a mild acidic environment (pH 6.0). When the environment is recycled to the neutral (pH 7.4) plasma membrane, the interaction is lost and the antibody is released back into the circulation. This affects the plasma half-life of IgG.

The ability of the IgG1-005 mutants E345K, E345Q, E345R, E345Y, E430F, E430G, E430S, E430T, S440Y, K439E and S440K to interact with FcRn from mice, cynomolgus monkeys and humans was tested in ELISA. Mutants P247G and I253D were also tested in mouse FcRn ELISA. I253D was used as a negative control for binding to FcRn. All incubations were performed at room temperature. A 96-well plate was coated with 5 μg/mL (100 μL/well) of the recombinantly produced biotinylated extracellular domain (FcRnECDHis-B2M-BIO) of FcRn (mouse, human or cynomolgus), and PBST Diluted in +0.2% BSA and incubated for 1 hour. Plates were washed 3 times with PBST, wild-type IgG1-005 or 005 mutants serially diluted 3 times (in PBST/0.2% BSA, pH 6.0) were added and the plates were incubated for 1 hour. Plates were washed with PBST/0.2% BSA, pH 6.0. Goat-anti-human IgG(Fab'2)-HRP (Jackson Immuno Research, Cat. No.: 109-035-097) diluted in PBST/0.2% BSA, pH 6.0 was added, and the plate was incubated for 1 hour. . After washing, ABTS was added as a substrate and the plate was incubated for 30 minutes in the dark. Absorbance was read at 405 nm using an EL808 ELISA reader. The data generated in mouse FcRn ELISA were analyzed using the best-fit value of a non-linear agonist dose-response fit using log-transformed concentrations in GraphPad Prism 5, and the apparent affinity EC ₅₀ was calculated ( Table 20). Experiments show that FcRn binding was not altered by any of the IgG1-005 mutants compared to wild-type IgG1-005.

[Table 20]

Figure 29 shows that all tested mutants of wild type IgG1-005 and IgG1-005 bind well to mouse, human and cynomolgus FcRn at pH 6.0. No significant binding to FcRn was detected at pH 7.4 (data not shown).

Example 35

CDC enhanced by different mutations of Rituximab in B cell lines Ramos and SU-DHL-4

As described in Example 19, oligomerization and CDC activity of anti-CD38 antibody IgG1-005 can be stimulated by single mutations at specific residues on or around the Fc:Fc interface. Oligomerization can also be stimulated indirectly by another type of mutation at residues away from the Fc:Fc interface that enhances the Fc:Fc interaction in an allosteric manner. It was also tested against the IgG1 anti-CD20 antibody rituximab in two B cell lines (Ramos and SU-DHL-4). The following mutations were tested: E345K, E345Q, E345R, E345Y, E430G, E430S, E430T, and S440Y (essentially as described in Example 19).

For the CDC assay, 0.1 x 10 ⁶ cells (Ramos or SU-DHL-4) were harvested in a round-bottom 96-well plate with 100 μL of saturated antibody at serial concentrations (0.0001-10.0 μg/mL in 3-fold dilution). Pre-incubate at RT on a shaker for 15 minutes in total volume. Subsequently, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated for 45 minutes in a 37° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS. Data were analyzed using the best-fit values of the non-linear agonist dose-response fit using log-transformed concentrations in GraphPad Prism 5. 30 shows that all tested rituximab mutants were able to increase CDC efficacy in both B-cells.

Example 36

Target independent fluid phase complement activation: IgG1-005 mutants were compared to wild-type IgG1-005

Target-independent complement activation can present safety issues when antibodies activate complement in bloodstream or organ tissue. This can lead to undesired complement activation products or undesired complement deposition. To test target-independent fluid phase complement activation, 100 μg/ml of IgG1-005 mutants E345K, E345Q, E345R, E345Y, E430F, E430G, E430S, E430T, S440Y, wild-type IgG1-005 or heat aggregated IgG (HAG , Positive control) were incubated at 37° C. for 1 hour in 90% normal human serum. The sample was then transferred to an ELISA-kit to measure C4d production (micro view C4d-fragment, Quidel, San Diego, CA, USA). C4d is an activating fragment of C4, a marker for classical complement pathway activation.

Figure 31 shows that wild-type IgG1-005, IgG1-005-E345K, IgG1-005-E345Q, IgG1-005-E345Y, IgG1-005-E430G, IgG1-005-E430S, and IgG1-005-S440Y showed minimal C4 activation. On the other hand, it shows that IgG1-005-E345R, IgG1-005-E430F and IgG1-005-E430T show increased C4d production (C4 activation) compared to wild-type IgG1-005.

Example 37

Plasma clearance rate of IgG1-005 mutant compared to wild-type IgG1-005

In this study, mice were housed in a barrier unit of a central laboratory animal facility (Utrecht, Netherlands) and maintained in Utrecht cages with unlimited provision of water and feed. All experiments were approved by the Utrecht University Animal Testing Ethics Committee. Using 3 mice per group, 500 μg antibody was intravenously injected into SCID mice (C.B-17/Icr-Prkdc<Scid>/IcrIcoCrl, Charles-River).

50 μL blood samples were collected from the saphenous vein 10 minutes, 4 hours, 1 day, 2 days, 7 days, 14 days and 21 days after antibody administration. Blood was collected in a vial containing heparin and centrifuged at 10,000 g for 5 minutes. Plasma was stored at -20°C until determination of antibody concentration.

Specific human IgG concentrations were determined using total hIgG and CD38 specific sandwich ELISA.

For total hIgG ELISA, a mouse mAb anti-human IgG-kappa clone MH16 (#M1268, CLB Sangquin (Netherlands) coated on a 96-well micron ELISA plate (Greiner, Germany) at a concentration of 2 μg/mL was captured antibody After the plate was blocked with PBS supplemented with 0.2% bovine serum albumin, samples were added, serially diluted in ELISA buffer (PBS supplemented with 0.05% Tween 20 and 0.2% bovine serum albumin), and plate shaken. Incubated on the bed for 1 hour at room temperature (RT) Plates were subsequently incubated with goat anti-human IgG immunoglobulin (#109-035-098, Jackson, West Grace, PA) and 2,2'- Color development was carried out with azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Roche, Mannheim, Germany) Absorbance was measured at 405 nm in a microplate reader (Biotech, Winuskey, Vermont).

For specific CD38 ELISA, His-tagged CD38 extracellular domains were coated on 96-well micron ELISA plates (Greiner, Germany) at a concentration of 2 μg/mL. After blocking the plate with ELISA buffer, samples serially diluted with ELISA buffer were added and incubated on a plate shaker for 1 hour at room temperature (RT). Plates were subsequently incubated with 30 ng/ml mouse anti-human IgG1-HRP (Sanquin M1328, clone MH161-1) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS ; Roche, Mannheim, Germany). The absorbance was measured at 405 nm in a microplate reader (Biotech, Winuskey, Vermont).

Figure 32a is a wild-type reference antibody IgG1-005 and antibody variants IgG1-005-E345K, IgG1-005-E345Q, IgG1-005-E345R, IgG1-005-E345Y, IgG1-005-E430F, IgG1-005-E430G, IgG1- It shows the IgG clearance rates of 005-E430S, IgG1-005-E430T, and IgG1-005-S440Y. Mutants IgG1-005-E430S, IgG1-005-E430G, and IgG1-005-S440Y, IgG1-005-E430T, IgG1-005-E345K, IgG1-005-E345Q, and IgG1-005-E345Y are wild-type IgG1-005 It showed a clearance rate similar to. Mutants IgG1-005-E430F and IgG1-005-E345R showed faster clearance rates. Plasma clearance rate was calculated as dose/AUC (mL/day/kg). The AUC value (area under the curve) was determined from the concentration-time curve.

Figure 32B shows the wild type reference antibody IgG1-005 and antibody variants IgG1-005-E345K, IgG1- as determined by CD38 specific ELISA when injected intravenously one day after intraperitoneal administration of 8.0 mg unrelated IgG1-B12 control antibody. The IgG clearance rates of 005-E345R, IgG1-005-E430G, IgG1-005-E430S, and IgG1-005-S440Y are shown. The wild type reference antibody IgG1 in the absence of an unrelated b12 control was included as a control. Mutants IgG1-005-E430S, IgG1-005-E430G, IgG1-005-S440Y and IgG1-005-E345K showed similar clearance rates as wild type. The mutant IgG1-005-E345R showed faster clearance.

Equivalent

One of ordinary skill in the art will recognize a number of equivalents to the specific embodiments of the invention described herein or can ascertain without using any further routine experimentation. These equivalents are intended to be covered by the following claims. Any and all combinations of the embodiments disclosed in the dependent claims are also contemplated as being within the scope of the present invention.

SEQUENCE LISTING <110> Genmab A/S <120> Polypeptide Variants and Uses Thereof <130> P/0077-WO <140> PC//EP2013/050429 <141> 2013-01-10 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 330 <212> PRT <213> homo sapiens <400> 1 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 2 <211> 326 <212> PRT <213> homo sapiens <400> 2 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 <210> 3 <211> 377 <212> PRT <213> homo sapiens <400> 3 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 <210> 4 <211> 327 <212> PRT <213> homo sapiens <400> 4 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 5 <211> 330 <212> PRT <213> homo sapiens <400> 5 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 6 <211> 201 <212> PRT <213> homo sapiens <400> 6 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 1 5 10 15 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 20 25 30 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 35 40 45 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 50 55 60 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 65 70 75 80 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 85 90 95 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 100 105 110 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 115 120 125 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 130 135 140 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 145 150 155 160 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 165 170 175 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 180 185 190 Lys Ser Leu Ser Leu Ser Pro Gly Lys 195 200 <210> 7 <211> 201 <212> PRT <213> homo sapiens <400> 7 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 1 5 10 15 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 20 25 30 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 35 40 45 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 50 55 60 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 65 70 75 80 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 85 90 95 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 100 105 110 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 115 120 125 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 130 135 140 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 145 150 155 160 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 165 170 175 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 180 185 190 Lys Ser Leu Ser Leu Ser Pro Gly Lys 195 200 <210> 8 <211> 201 <212> PRT <213> homo sapiens <400> 8 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 1 5 10 15 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 20 25 30 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 35 40 45 Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His 50 55 60 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 65 70 75 80 Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 85 90 95 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 100 105 110 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 115 120 125 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 130 135 140 Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 145 150 155 160 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 165 170 175 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 180 185 190 Lys Ser Leu Ser Leu Ser Pro Gly Lys 195 200 <210> 9 <211> 201 <212> PRT <213> homo sapiens <400> 9 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 1 5 10 15 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 20 25 30 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 35 40 45 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 50 55 60 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 65 70 75 80 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 85 90 95 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 100 105 110 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 115 120 125 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 130 135 140 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 145 150 155 160 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 165 170 175 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 180 185 190 Lys Ser Leu Ser Leu Ser Pro Gly Lys 195 200 <210> 10 <211> 201 <212> PRT <213> homo sapiens <400> 10 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 1 5 10 15 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 20 25 30 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 35 40 45 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 50 55 60 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 65 70 75 80 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 85 90 95 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 100 105 110 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 115 120 125 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 130 135 140 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 145 150 155 160 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 165 170 175 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 180 185 190 Lys Ser Leu Ser Leu Ser Leu Gly Lys 195 200 <210> 11 <211> 204 <212> PRT <213> homo sapiens <400> 11 Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile Thr Cys Leu 1 5 10 15 Val Val Asp Leu Ala Pro Ser Lys Gly Thr Val Asn Leu Thr Trp Ser 20 25 30 Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg Lys Glu Glu Lys 35 40 45 Gln Arg Asn Gly Thr Leu Thr Val Thr Ser Thr Leu Pro Val Gly Thr 50 55 60 Arg Asp Trp Ile Glu Gly Glu Thr Tyr Gln Cys Arg Val Thr His Pro 65 70 75 80 His Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Thr Ser Gly Pro 85 90 95 Arg Ala Ala Pro Glu Val Tyr Ala Phe Ala Thr Pro Glu Trp Pro Gly 100 105 110 Ser Arg Asp Lys Arg Thr Leu Ala Cys Leu Ile Gln Asn Phe Met Pro 115 120 125 Glu Asp Ile Ser Val Gln Trp Leu His Asn Glu Val Gln Leu Pro Asp 130 135 140 Ala Arg His Ser Thr Thr Gln Pro Arg Lys Thr Lys Gly Ser Gly Phe 145 150 155 160 Phe Val Phe Ser Arg Leu Glu Val Thr Arg Ala Glu Trp Glu Gln Lys 165 170 175 Asp Glu Phe Ile Cys Arg Ala Val His Glu Ala Ala Ser Pro Ser Gln 180 185 190 Thr Val Gln Arg Ala Val Ser Val Asn Pro Gly Lys 195 200 <210> 12 <211> 221 <212> PRT <213> homo sapiens <400> 12 Ala Leu Glu Asp Leu Leu Leu Gly Ser Glu Ala Asn Leu Thr Cys Thr 1 5 10 15 Leu Thr Gly Leu Arg Asp Ala Ser Gly Val Thr Phe Thr Trp Thr Pro 20 25 30 Ser Ser Gly Lys Ser Ala Val Gln Gly Pro Pro Glu Arg Asp Leu Cys 35 40 45 Gly Cys Tyr Ser Val Ser Ser Val Leu Pro Gly Cys Ala Glu Pro Trp 50 55 60 Asn His Gly Lys Thr Phe Thr Cys Thr Ala Ala Tyr Pro Glu Ser Lys 65 70 75 80 Thr Pro Leu Thr Ala Thr Leu Ser Lys Ser Gly Asn Thr Phe Arg Pro 85 90 95 Glu Val His Leu Leu Pro Pro Pro Ser Glu Glu Leu Ala Leu Asn Glu 100 105 110 Leu Val Thr Leu Thr Cys Leu Ala Arg Gly Phe Ser Pro Lys Asp Val 115 120 125 Leu Val Arg Trp Leu Gln Gly Ser Gln Glu Leu Pro Arg Glu Lys Tyr 130 135 140 Leu Thr Trp Ala Ser Arg Gln Glu Pro Ser Gln Gly Thr Thr Thr Phe 145 150 155 160 Ala Val Thr Ser Ile Leu Arg Val Ala Ala Glu Asp Trp Lys Lys Gly 165 170 175 Asp Thr Phe Ser Cys Met Val Gly His Glu Ala Leu Pro Leu Ala Phe 180 185 190 Thr Gln Lys Thr Ile Asp Arg Leu Ala Gly Lys Pro Thr His Val Asn 195 200 205 Val Ser Val Val Met Ala Glu Val Asp Gly Thr Cys Tyr 210 215 220 <210> 13 <211> 221 <212> PRT <213> homo sapiens <400> 13 Ala Leu Glu Asp Leu Leu Leu Gly Ser Glu Ala Asn Leu Thr Cys Thr 1 5 10 15 Leu Thr Gly Leu Arg Asp Ala Ser Gly Ala Thr Phe Thr Trp Thr Pro 20 25 30 Ser Ser Gly Lys Ser Ala Val Gln Gly Pro Pro Glu Arg Asp Leu Cys 35 40 45 Gly Cys Tyr Ser Val Ser Ser Val Leu Pro Gly Cys Ala Gln Pro Trp 50 55 60 Asn His Gly Glu Thr Phe Thr Cys Thr Ala Ala His Pro Glu Leu Lys 65 70 75 80 Thr Pro Leu Thr Ala Asn Ile Thr Lys Ser Gly Asn Thr Phe Arg Pro 85 90 95 Glu Val His Leu Leu Pro Pro Pro Ser Glu Glu Leu Ala Leu Asn Glu 100 105 110 Leu Val Thr Leu Thr Cys Leu Ala Arg Gly Phe Ser Pro Lys Asp Val 115 120 125 Leu Val Arg Trp Leu Gln Gly Ser Gln Glu Leu Pro Arg Glu Lys Tyr 130 135 140 Leu Thr Trp Ala Ser Arg Gln Glu Pro Ser Gln Gly Thr Thr Thr Phe 145 150 155 160 Ala Val Thr Ser Ile Leu Arg Val Ala Ala Glu Asp Trp Lys Lys Gly 165 170 175 Asp Thr Phe Ser Cys Met Val Gly His Glu Ala Leu Pro Leu Ala Phe 180 185 190 Thr Gln Lys Thr Ile Asp Arg Leu Ala Gly Lys Pro Thr His Val Asn 195 200 205 Val Ser Val Val Met Ala Glu Val Asp Gly Thr Cys Tyr 210 215 220 <210> 14 <211> 223 <212> PRT <213> homo sapiens <400> 14 Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser Thr Lys Leu Thr Cys Leu 1 5 10 15 Val Thr Asp Leu Thr Thr Tyr Asp Ser Val Thr Ile Ser Trp Thr Arg 20 25 30 Gln Asn Gly Glu Ala Val Lys Thr His Thr Asn Ile Ser Glu Ser His 35 40 45 Pro Asn Ala Thr Phe Ser Ala Val Gly Glu Ala Ser Ile Cys Glu Asp 50 55 60 Asp Trp Asn Ser Gly Glu Arg Phe Thr Cys Thr Val Thr His Thr Asp 65 70 75 80 Leu Pro Ser Pro Leu Lys Gln Thr Ile Ser Arg Pro Lys Gly Val Ala 85 90 95 Leu His Arg Pro Asp Val Tyr Leu Leu Pro Pro Ala Arg Glu Gln Leu 100 105 110 Asn Leu Arg Glu Ser Ala Thr Ile Thr Cys Leu Val Thr Gly Phe Ser 115 120 125 Pro Ala Asp Val Phe Val Gln Trp Met Gln Arg Gly Gln Pro Leu Ser 130 135 140 Pro Glu Lys Tyr Val Thr Ser Ala Pro Met Pro Glu Pro Gln Ala Pro 145 150 155 160 Gly Arg Tyr Phe Ala His Ser Ile Leu Thr Val Ser Glu Glu Glu Trp 165 170 175 Asn Thr Gly Glu Thr Tyr Thr Cys Val Ala His Glu Ala Leu Pro Asn 180 185 190 Arg Val Thr Glu Arg Thr Val Asp Lys Ser Thr Gly Lys Pro Thr Leu 195 200 205 Tyr Asn Val Ser Leu Val Met Ser Asp Thr Ala Gly Thr Cys Tyr 210 215 220 <210> 15 <211> 209 <212> PRT <213> homo sapiens <400> 15 Ala Val Gln Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe 1 5 10 15 Val Val Gly Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala 20 25 30 Gly Lys Val Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His 35 40 45 Ser Asn Gly Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser 50 55 60 Leu Trp Asn Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser 65 70 75 80 Leu Pro Pro Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala 85 90 95 Pro Val Lys Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu 100 105 110 Ala Ala Ser Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn 115 120 125 Ile Leu Leu Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly 130 135 140 Phe Ala Pro Ala Arg Pro Pro Pro Gln Pro Gly Ser Thr Thr Phe Trp 145 150 155 160 Ala Trp Ser Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala 165 170 175 Thr Tyr Thr Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn 180 185 190 Ala Ser Arg Ser Leu Glu Val Ser Tyr Val Thr Asp His Gly Pro Met 195 200 205 Lys <210> 16 <211> 454 <212> PRT <213> homo sapiens <400> 16 Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr Gly 20 25 30 Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 35 40 45 Val Ile Trp Asp Asp Gly Ser Tyr Lys Tyr Tyr Gly Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Gly Ile Thr Met Val Arg Gly Val Met Lys Asp Tyr Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445 Ser Leu Ser Pro Gly Lys 450 <210> 17 <211> 451 <212> PRT <213> homo sapiens <400> 17 Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr Gly 20 25 30 Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 35 40 45 Val Ile Trp Asp Asp Gly Ser Tyr Lys Tyr Tyr Gly Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Gly Ile Thr Met Val Arg Gly Val Met Lys Asp Tyr Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser 210 215 220 Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 260 265 270 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Leu Gly Lys 450 <210> 18 <211> 97 <212> PRT <213> homo sapiens <400> 18 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 1 5 10 15 Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys Pro 20 25 30 Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu 35 40 45 Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro 50 55 60 Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala Pro Glu 65 70 75 80 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 85 90 95 Thr

Claims (1)

Comprising introducing a mutation at one or more amino acid residue(s) selected from the group corresponding to E430X, E345X, and S440W in the Fc region of a human IgG1 heavy chain into a parent polypeptide comprising the Fc domain and binding region of an immunoglobulin. , A method of increasing the complement-dependent cytotoxicity (CDC) of said parental polypeptide.

Images